Hepatitis C virus protease

ABSTRACT

The protease necessary for polyprotein processing in Hepatitis C virus is identified, cloned, and expressed. Proteases, truncated protease, and altered proteases are disclosed which are useful for cleavage of specific polypeptides, and for assay and design of antiviral agents specific for HCV.

This application is a divisional, of application Ser. No. 08/350,884, filed Dec. 6, 1994, which is a divisional of application Ser. No. 07/680,296, filed Apr. 4, 1991, now U.S. Pat. No. 5,371,017.

TECHNICAL FIELD

This invention relates to the molecular biology and virology of the hepatitis C virus (HCV). More specifically, this invention relates to a novel protease produced by HCV, methods of expression, recombinant protease, protease mutants, and inhibitors of HCV protease.

BACKGROUND OF THE INVENTION

Non-A, Non-B hepatitis (NANBH) is a transmissible disease (or family of diseases) that is believed to be vitally induced, and is distinguishable from other forms of virus-associated liver disease, such as those caused by hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Epidemiologic evidence suggests that there may be three types of NANBH: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring (community acquired) type. However, the number of causative agents is unknown. Recently, however, a new viral species, hepatitis C virus (HCV) has been identified as the primary (if not only) cause of blood-associated NANBH (BB-NANBH). See for example, PCT WO89/046699; U.S. patent application Ser. No. 7/456,637, filed 21 Dec. 1989; and U.S. patent application Ser. No. 7/456,637, filed 21 Dec. 1989, incorporated herein by reference. Hepatitis C appears to be the major form of transfusion-associated hepatitis in a number of countries, including the United States and Japan. There is also evidence implicating HCV in induction of hepatocellular carcinoma. Thus, a need exists for an effective method for treating HCV infection: currently, there is none.

Many viruses, including adenovimses, baculoviruses, comoviruses, picomaviruses, retroviruses, and togaviruses, rely on specific, virally-encoded proteases for processing polypeptides from their initial translated form into mature, active proteins. In the case of picomaviruses, all of the viral proteins are believed to arise from cleavage of a single polyprotein (B. D. Korant, CRC Crit Rev Biotech (1988) 8:149-57).

S. Pichuantes et al, in "Viral Proteinases As Targets For Chemotherapy" (Cold Spring Harbor Laboratory Press, 1989) pp. 215-22, disclosed expression of a vital protease found in HIV-1. The HIV protease was obtained in the form of a fusion protein, by fusing DNA encoding an HIV protease precursor to DNA encoding human superoxide dismutase (hSOD), and expressing the product in E. coli. Transformed cells expressed products of 36 and 10 kDa (corresponding to the hSOD-protease fusion protein and the protease alone), suggesting that the protease was expressed in a form capable of autocatalytic proteolysis.

T. J. McQuade et al, Science (1990) 247:454-56 disclosed preparation of a peptide mimic capable of specifically inhibiting the HIV-1 protease. In HIV, the protease is believed responsible for cleavage of the initial p55 gag precursor transcript into the core structural proteins (p 17, p24, p8, and p7). Adding 1 μM inhibitor to HIV-infected peripheral blood lymphocytes in culture reduced the concentration of processed HIV p24 by about 70%. Viral maturation and levels of infectious virus were reduced by the protease inhibitor.

DISCLOSURE OF THE INVENTION

We have now invented recombinant HCV protease, HCV protease fusion proteins, truncated and altered HCV proteases, cloning and expression vectors therefore, and methods for identifying antiviral agents effective for treating HCV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence of HCV protease (SEQ ID NO:69 and SEQ ID NO:70).

FIG. 2 shows the polynucleotide sequence and deduced amino acid sequence of the clone C20c (SEQ ID NO:71 and SEQ ID NO:72).

FIG. 3 shows the polynucleotide sequence and deduced amino acid sequence of the clone C26d (SEQ ID NO:73 and SEQ ID NO:74).

FIG. 4 shows the polynucleotide sequence and deduced amino acid sequence of the clone C8h (SEQ ID NO:75 and SEQ ID NO:76).

FIG. 5 shows the polynucleotide sequence and deduced amino acid sequence of the clone C7f (SEQ ID NO:77 and SEQ ID NO:78).

FIG. 6 shows the polynucleotide sequence and deduced amino acid sequence of the clone C31 (SEQ ID NO:79 and SEQ ID NO:80).

FIG. 7 shows the polynucleotide sequence and deduced amino acid sequence of the clone C35 (SEQ ID NO:81 and SEQ ID NO:82).

FIG. 8 shows the polynucleotide sequence and deduced amino acid sequence of the clone C33c (SEQ ID NO:83 and SEQ ID NO:84).

FIG. 9 schematically illustrates assembly of the vector C7fC20cC300C200.

FIG. 10 shows the sequence of vector cf1SODp600 (SEQ ID NO:85 and SEQ ID NO:86).

MODES OF CARRYING OUT THE INVENTION A. Definitions

The terms "Hepatitis C Virus" and "HCV" refer to the vital species that is the major etiological agent of BB-NANBH, the prototype isolate of which is identified in PCT WO89/046699; EPO publication 318,216; U.S. Ser. No. 7/355,008, filed 18 May 1989; and U.S. Ser. No. 7/456,637, the disclosures of which are incorporated herein by reference. "HCV" as used herein includes the pathogenic strains capable of causing hepatitis C, and attenuated strains or defective interfering particles derived therefrom. The HCV genome is comprised of RNA. It is known that RNA-containing viruses have relatively high rates of spontaneous mutation, reportedly on the order of 10⁻³ to 10⁻⁴ per incorporated nucleotide (Fields & Knipe, "Fundamental Virology" (1986, Raven Press, N.Y.)). As heterogeneity and fluidity of genotype are inherent characteristics of RNA viruses, there will be multiple strains/isolates, which may be virulent or avirulent, within the HCV species.

Information on several different strains/isolates of HCV is disclosed herein, particularly strain or isolate CDC/HCVI (also called HCV1). Information from one strain or isolate, such as a partial genomic sequence, is sufficient to allow those skilled in the art using standard techniques to isolate new strains/isolates and to identify whether such new strains/isolates are HCV. For example, several different strains/isolates are described below. These strains, which were obtained from a number of human sero (and from different geographical areas), were isolated utilizing the information from the genomic sequence of HCV1.

The information provided herein suggests that HCV may be distantly related to the flaviviriclae. The Flavivirus family contains a large number of viruses which are small, enveloped pathogens of man. The morphology and composition of Flavivirus particles are known, and are discussed in M. A. Brinton, in "The Viruses: The Togaviridae And Flaviviridae" (Series eds. Fraenkel-Conrat and Wagner, vol. eds. Schlesinger and Schlesinger, Plenum Press, 1986), pp. 327-374. Generally, with respect to morphology, Flaviviruses contain a central nucleocapsid surrounded by a lipid bilayer. Virions are spherical and have a diameter of about 40-50 nm. Their cores are about 25-30 nm in diameter. Along the outer surface of the virion envelope are projections measuring about 5-10 nm in length with terminal knobs about 2 nm in diameter. Typical examples of the family include Yellow Fever virus, West Nile virus, and Dengue Fever virus. They possess positive-stranded RNA genomes (about 11,000 nucleotides) that are slightly larger than that of HCV and encode a polyprotein precursor of about 3500 amino acids. Individual viral proteins are cleaved from this precursor polypeptide.

The genome of HCV appears to be single-stranded RNA containing about 10,000 nucleotides. The genome is positive-stranded, and possesses a continuous translational open reading frame (ORF) that encodes a polyprotein of about 3,000 amino acids. In the ORF, the structural proteins appear to be encoded in approximately the first quarter of the N-terminal region, with the majority of the polyprotein attributed to non-structural proteins. When compared with all known viral sequences, small but significant co-linear homologies are observed with the non-structural proteins of the Flavivirus family, and with the pestiviruses (which are now also considered to be pan of the Flavivirus family).

A schematic alignment of possible regions of a flaviviral polyprotein (using Yellow Fever Virus as an example), and of a putative polyprotein encoded in the major ORF of the HCV genome,.is shown in FIG. 1. Possible domains of the HCV polyprotein are indicated in the figure. The Yellow Fever Virus polyprotein contains, from the amino terminus to the carboxy terminus, the nucleocapsid protein (C), the matrix protein (M), the envelope protein (E), and the non-structural proteins 1, 2 (a+b), 3, 4 (a+b), and 5 (NS1, NS2, NS3, NS4, and NS5). Based upon the putative amino acids encoded in the nucleotide sequence of HCV1, a small domain at the extreme N-terminus of the HCV polyprotein appears similar both in size and high content of basic residues to the nucleocapsid protein (C) found at the N-terminus of flaviviral polyproteins. The non-structural proteins 2,3,4, and 5 (NS2-5) of HCV and of yellow fever virus (YFV) appear to have counterparks of similar size and hydropathicity, although the amino acid sequences diverge. However, the region of HCV which would correspond to the regions of YFV polyprotein which contains the M, E, and NS1 protein not only differs in sequence, but also appears to be quite different in size and hydropathicity. Thus, while certain domains of the HCV genome may be referred to herein as, for example, NS1, or NS2, it should be understood that these designations are for convenience of reference only; there may be considerable differences between the HCV family and flaviviruses that have yet to be appreciated.

Due to the evolutionary relationship of the strains or isolates of HCV, putative HCV strains and isolates are identifiable by their hornology at the polypeptide level. With respect to the isolates disclosed herein, new HCV strains or isolates are expected to be at least about 40% homologous, some more than about 70% homologous, and some even more than about 80% homologous: some may be more than about 90% homologous at the polypeptide level. The techniques for determining amino acid sequence hornology are known in the art. For example, the amino acid sequence may be determined directly and compared to the sequences provided herein. Alternatively the nucleotide sequence of the genomic material of the putative HCV may be determined (usually via a cDNA intermediate), the amino acid sequence encoded therein can be determined, and the corresponding regions compared.

The term "HCV protease" refers to an enzyme derived from HCV which exhibits proteolytic activity, specifically the polypeptide encoded in the NS3 domain of the HCV genome. At least one strain of HCV contains a protease believed to be substantially encoded by or within the following sequence:

    ______________________________________     Arg Arg Gly Arg Glu Ile Leu Leu Gly Pro                              10     Ala Asp Gly Met Val Ser Lys Gly Trp Arg                              20     Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln                              30     Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile                              40     Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln                              50     Val Glu Gly Glu Val Gln Ile Val Ser Thr                              60     Ala Ala Gln Thr Phe Leu Ala Thr Cys Ile                              70     Asn Gly Val Cys Trp Thr Val Tyr His Gly                              80     Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys                              90     Gly Pro Val Ile Gln Met Tyr Thr Asn Val                              100     Asp Gln Asp Leu Val Gly Trp Pro Ala Ser                              110     Gln Gly Thr Arg Ser Leu Thr Pro Cys Thr                              120     Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr                              130     Arg His Ala Asp Val Ile Pro Val Arg Arg                              140     Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser                              150     Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser                              160     Ser Gly Gly Pro Leu Leu Cys Pro Ala Gly                              170     His Ala Val Gly Ile Phe Arg Ala Ala Val                              180     Cys Thr Arg Gly Val Ala Lys Ala Val Asp                              190     Phe Ile Pro Val Glu Asn Leu Glu Thr Thr                              200     Met Arg (SEQ ID NO: 1)...                              202     ______________________________________

The above N and C termini are putative, the actual termini being defined by expression and processing in an appropriate host of a DNA construct encoding the entire NS3 domain. It is understood that this sequence may vary from strain to strain, as RNA viruses like HCV are known to exhibit a great deal of variation. Further, the actual N and C termini may vary, as the protease is cleaved from a precursor polyprotein: variations in the protease amino acid sequence can result in cleavage from the polyprotein at different points. Thus, the amino- and carboxy-termini may differ from strain to strain of HCV. The first amino acid shown above corresponds to residue 60 in FIG. 1. However, the minimum sequence necessary for activity can be determined by routine methods. The sequence may be truncated at either end by treating an appropriate expression vector with an exonuclease (after cleavage at the 5' or 3' end of the coding sequence) to remove any desired number of base pairs. The resulting coding polynucleotide is then expressed and the sequence determined. In this manner the activity of the resulting product may be correlated with the amino acid sequence: a limited series of such experiments (removing progressively greater numbers of base pairs) determines the minimum internal sequence necessary for protease activity. We have found that the sequence may be substantially truncated, particularly at the carboxy terminus, apparently with full retention of protease activity. It is presently believed that a portion of the protein at the carboxy terminus may exhibit helicase activity. However, helicase activity is not required of the HCV proteases of the invention. The amino terminus may also be truncated to a degree without loss of protease activity.

The amino acids underlined above are believed to be the residues necessary for catalytic activity, based on sequence hornology to putative flavivirus serine proteases. Table 1 shows the alignment of the three serine protease catalytic residues for HCV protease and the protease obtained from Yellow-Fever Virus, West Nile Fever virus, Murray Valley Fever virus, and Kunjin virus. Although the other four flavivirus protease sequences exhibit higher homology with each other than with HCV, a degree of hornology is still observed with HCV. This hornology, however, was not sufficient for indication by currently available alignment software. The indicated amino acids are numbered His₇₉, Asp₁₀₃, and Ser₁₆₁ in the sequence listed above (His₁₃₉, Asp163, and Ser₂₂₂ in FIG. 1).

                                      TABLE 1     __________________________________________________________________________     Alignment of Active Residues by Sequence     Protease  His               Asp               Ser     __________________________________________________________________________     HCV       CWTVY HGAG (SEQ ID NO. 2)                                 DQ DLGWPAP (SEQ ID NO: 3)                                                   LKGS SGGPL (SEQ ID NO: 4)     Yellow Fever               FHTMW HVTR (SEQ ID NO. 5)                                 KE DLVAYGG (SEQ ID NO: 6)                                                   PSGT SGSPI (SEQ ID NO: 7)     West Nile Fever               FHTLW HTTK (SEQ ID NO. 8)                                 KE DRLCYGG (SEQ ID NO: 9)                                                   PTGT SGSPI (SEQ ID NO: 10)     Murray Valley               FHTLW HTTR (SEQ ID NO. 11)                                 KE DRVTYGG (SEQ ID NO: 12)                                                   PIGT SGSPI (SEQ ID NO: 13)     Kunjin Virus               FHTLW HTTK (SEQ ID NO. 14)                                 KE DRLCYGG (SEQ ID NO: 15)                                                   PTGT SGSPI (SEQ ID NO:     __________________________________________________________________________                                                   16)

Alternatively, one can make catalytic residue assignments based on structural homology. Table 2 shows alignment of HCV with against the catalytic sites of several well-characterized serine proteases based on structural considerations: protease A from Streptomyces griseus, α-lytic protease, bovine trypsin, chymotrypsin, and elastase (M. James et al, Can J Biochem (1978) 56:396). Again, a degree of homology is observed. The HCV residues identified are numbered His₇₉, Asp₁₂₅, and Ser₁₆₁ in the sequence listed above.

                                      TABLE 2     __________________________________________________________________________     Alignment of Active Residues by Structure     Protease His           Asp             Ser     __________________________________________________________________________     S. griseus A              TAG HC (SEQ ID NO: 17)                            NN DYGII (SEQ ID NO: 18)                                            GD SGGSL (SEQ ID NO: 19)     α-Lytic protease              TAG HC (SEQ ID NO: 20)                            GN DRAWV (SEQ ID NO: 21)                                            GD SGGSW (SEQ ID NO: 22)     Bovine Trypsin              SAA HC (SEQ ID NO: 23)                            NN DIMLI (SEQ ID NO: 24)                                            GD SGGPV (SEQ ID NO: 25)     Chymotrypsin              TAA HC (SEQ ID NO: 26)                            NN DITLL (SEQ ID NO: 27)                                            GD SGGPL (SEQ ID NO: 28)     Elastase TAA HC (SEQ ID NO: 29)                            GY DIALL (SEQ ID NO: 30)                                            GD SGGPL (SEQ ID NO: 31)     HCV      TVY HG (SEQ ID NO: 32)                            SS DLYLV (SEQ ID NO: 33)                                            GS SGGPL (SEQ ID NO:     __________________________________________________________________________                                            34)

The most direct manner to verify the residues essential to the active site is to replace each residue individually with a residue of equivalent stearic size. This is easily accomplished by site-specific mutagenesis and similar methods known in the art. If replacement of a particular residue with a residue of equivalent size results in loss of activity, the essential nature of the replaced residue is confirmed.

"HCV protease analogs" refer to polypeptides which vary from the full length protease sequence by deletion, alteration and/or addition to the amino acid sequence of the native protease. HCV protease analogs include the truncated proteases described above, as well as HCV protease muteins and fusion proteins comprising HCV protease, truncated protease, or protease muteins. Alterations to form HCV protease muteins are preferably conservative amino acid substitutions, in which an amino acid is replaced with another naturally-occurring amino acid of similar character. For example, the following substitutions are considered "conservative":______________________________________Gly ←→ Ala; Asp ←→ Glu;Val ←→ Ile ←→ Leu;Lys ←→ Arg;Asn ←→ Gln; andPhe ←→ Trp ←→ Tyr.______________________________________

Nonconservative changes are generally substitutions of one of the above amino acids with an amino acid from a different group (e.g., substituting Asn for Glu), or substituting Cys, Met, His, or Pro for any of the above amino acids. Substitutions involving common amino acids are conveniently performed by site specific mutagenesis of an expression vector encoding the desired protein, and subsequent expression of the altered form. One may also alter amino acids by synthetic or semi-synthetic methods. For example, one may convert cysteine or serine residues to selenocysteine by appropriate chemical treatment of the isolated protein. Alternatively, one may incorporate uncommon amino acids in standard in vitro protein synthetic methods. Typically, the total number of residues changed, deleted or added to the native sequence in the muteins will be no more than about 20, preferably no more than about 10, and most preferably no more than about 5.

The term fusion protein generally refers to a polypeptide comprising an amino acid sequence drawn from two or more individual proteins. In the present invention, "fusion protein" is used to denote a polypeptide comprising the HCV protease, truncate, mutein or a functional portion thereof, fused to a non-HCV protein or polypeptide ("fusion partner"). Fusion proteins are most conveniently produced by expression of a fused gene, which encodes a portion of one polypeptide at the 5' end and a portion of a different polypeptide at the 3' end, where the different portions are joined in one reading frame which may be expressed in a suitable host. It is presently preferred (although not required) to position the HCV protease or analog at the carboxy terminus of the fusion protein, and to employ a functional enzyme fragment at the amino terminus. As the HCV protease is normally expressed within a large polyprotein, it is not expected to include cell transport signals (e.g., export or secretion signals). Suitable functional enzyme fragments are those polypeptides which exhibit a quantifiable activity when expressed fused to the HCV protease. Exemplary enzymes include, without limitation, α-galactosidase (α-gal), α-lactamase, horseradish peroxidase (HRP), glucose oxidase (GO), human superoxide dismutase (hSOD), urease, and the like. These enzymes are convenient because the amount of fusion protein produced can be quantified by means of simple colorimetric assays. Alternatively, one may employ antigenic proteins or fragments, to permit simple detection and quantification of fusion proteins using antibodies specific for the fusion partner. The presently preferred fusion partner is hSOD.

B. General Method

The practice of the present invention generally employs conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See for example J. Sambrook et al, "Molecular Cloning; A Laboratory Manual (1989); "DNA Cloning", Vol. I and II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed, 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins eds. 1984); "Transcription And Translation" (B. D. Hames & S. J. Higgins eds. 1984); "Animal Cell Culture" (R. I. Freshney ed. 1986); "Immobilized Cells And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984); the series, "Methods In Enzymology" (Academic Press, Inc.); "Gene Transfer Vectors For Mammalian Cells" (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Meth Enzymol (1987) 154 and 155 (Wu and Grossman, and Wu, eds., respectively); Mayer & Walker, eds. (1987), "Immunochemical Methods In Cell And Molecular Biology" (Academic Press, London); Scopes, "Protein Purification: Principles And Practice", 2nd Ed (Springer-Verlag, N.Y., 1987); and "Handbook Of Experimental Immunology", volumes I-IV (Weir and Blackwell, eds, 1986).

Both prokaryotic and eukaryotic host cells are useful for expressing desired coding sequences when appropriate control sequences compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers. These plasmids are commercially available. The markers may be used to obtain successful transformants by selection. Commonly used prokaryotic control sequences include the ε-lactamase (penicillinase) and lactose promoter systems (Chang et al, Nature (1977) 198:1056), the tryptophan (trp) promoter system (Goeddel et al, Nuc Acids Res (1980) 8:4057) and the lambda-derived P_(L) promoter and N gene ribosome binding site (Shimatake et al, Nature (1981) 292:128) and the hybrid tac promoter (De Boer et al, Proc Nat Acad Sci USA (1983) 292:128) derived from sequences of the trp and lac UV5 promoters. The foregoing systems are particularly compatible with E. coli; if desired, other prokaryotic hosts such as strains of Bacillus or Pseudomonas may be used, with corresponding control sequences.

Eukaryotic hosts include without limitation yeast and mammalian cells in culture systems. Yeast expression hosts include Saccharomyces, Klebsiella, Picia, and the like. Saccharomyces cerevisiae and Saccharomyces carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts. Yeast-compatible vectors carry markers which permit selection of successful transformants by conferring prototrophy to auxotrophic mutants or resistance to heavy metals on wild-type strains. Yeast compatible vectors may employ the 2 μ origin of replication (Broach et al, Meth Enzymol (1983) 101:307), the combination of CEN3 and ARS1 or other means for assuring replication, such as sequences which will result in incorporation of an appropriate fragment into the host cell genome. Control sequences for yeast vectors are known in the art and include promoters for the synthesis of glycolytic enzymes (Hess et al, J Adv Enzyme Reg (1968) 7:149; Holland et al, Biochem (1978), 17:4900), including the promoter for 3-phosphoglycerate kinase (R. Hitzeman et al, J Biol Chem (1980) 255:2073). Terminators may also be included, such as those derived from the enolase gene (Holland, J Biol Chem (1981) 256:1385). Particularly useful control systems are those which comprise the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) promoter or alcohol dehydrogenase (ADH) regulatable promoter, terminators also derived from GAPDH, and if secretion is desired, a leader sequence derived from yeast α-factor (see U.S. Pat. No. 4,870,008, incorporated herein by reference).

A presently preferred expression system employs the ubiquitin leader as the fusion partner. Copending application U.S. Ser. No. 7/390,599 filed 7 Aug. 1989 disclosed vectors for high expression of yeast ubiquitin fusion proteins. Yeast ubiquitin provides a 76 amino acid polypeptide which is automatically cleaved from the fused protein upon expression. The ubiquitin amino acid sequence is as follows:__________________________________________________________________________Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr (SEQ ID NO: 35)Leu Glu Val Glu Ser Ser Asp Thr Ile Asp Asn Val LysSer Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp GlnGln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp GlyArg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser ThrLeu His Leu Val Leu Arg Leu Arg Gly Gly__________________________________________________________________________

See also Ozkaynak et at, Nature (1984) 312:663-66. Polynucleotides encoding the ubiquitin polypeptide may be synthesized by standard methods, for example following the technique of Barr et al, J Biol Chem (1988) 268:1671-78 using an Applied Biosystem 380A DNA synthesizer. Using appropriate linkers, the ubiquitin gene may be inserted into a suitable vector and ligated to a sequence encoding the HCV protease or a fragment thereof.

In addition, the transcriptional regulatory region and the transcriptional initiation region which are operably linked may be such that they are not naturally associated in the wild-type organism. These systems are described in detail in EPO 120,551, published October 3, 1984; EPO 116,201, published Aug. 22, 1984; and EPO 164,556, published Dec. 18, 1985, all of which are commonly owned with the present invention, and are hereby incorporated herein by reference in full.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and a number of other cell lines. Suitable promoters for mammalian cells are also known in the art and include vital promoters such as that from Simian Virus 40 (SV40) (Fiers et al, Nature (1978) 273:113), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammalian cells may also require terminator sequences and poly-A addition sequences. Enhancer sequences which increase expression may also be included, and sequences which promote amplification of the gene may also be desirable (for example methotrexate resistance genes). These sequences are known in the art.

Vectors suitable for replication in mammalian cells are known in the art, and may include vital replicons, or sequences which insure integration of the appropriate sequences encoding HCV epitopes into the host genome. For example, another vector used to express foreign DNA is Vaccinia virus. In this case the heterologous DNA is inserted into the Vaccinia genome. Techniques for the insertion of foreign DNA into the vaccinia virus genome are known in the art, and may utilize, for example, homologous recombination. The heterologous DNA is generally inserted into a gene which is non-essential to the virus, for example, the thyroidinc kinase gene (tk), which also provides a selectable marker. Plasmid vectors that greatly facilitate the construction of recombinant viruses have been described (see, for example, Mackett et al, J Virol (1984) 49:857; Chakrabarti et al, Mol Cell Biol (1985) 5:3403; Moss, in GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (Miller and Calos, eds., Cold Spring Harbor Laboratory, N.Y., 1987), p. 10). Expression of the HCV polypeptide then occurs in cells or animals which are infected with the live recombinant vaccinia virus.

In order to detect whether or not the HCV polypeptide is expressed from the vaccinia vector, BSC 1 cells may be infected with the recombinant vector and grown on microscope slides under conditions which allow expression. The cells may then be acetone-fixed, and immunofluorescence assays performed using serum which is known to contain anti-HCV antibodies to a polypeptide(s) encoded in the region of the HCV genome from which the HCV segment in the recombinant expression vector was derived.

Other systems for expression of eukaryotic or vital genomes include insect cells and vectors suitable for use in these cells. These systems are known in the art, and include, for example, insect expression transfer vectors derived from the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), which is a helper-independent, viral expression vector. Expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive expression of heterologous genes. Currently the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373 (see PCT WO89/046699 and U.S. Ser. No. 7/456,637). Many other vectors known to those of skill in the an have also been designed for improved expression. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and introduces a BamHI cloning site 32 bp downstream from the ATT; See Luckow and Summers, Virol (1989) 17:31). AcNPV transfer vectors for high level expression of nonfused foreign proteins are described in copending applications PCT WO89/046699 and U.S. Ser. No. 7/456,637. A unique BamHI site is located following position -8 with respect to the translation initiation codon ATG of the polyhedrin gene. There are no cleavage sites for SmaI, PstI, BglII, XbaI or SstI. Good expression of nonfused foreign proteins usually requires foreign genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. The plasmid also contains the polyhedrin polyadenylation signal and the ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.

Methods for the introduction Of heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summer and Smith, Texas Agricultural Experiment Station Bulletin No. 1555; Smith et al, Mol Cell Biol (1983) 3:2156-2165; and Luckow and Summers, Virol (1989) 17:31). For example, the heterologous DNA can be inserted into a gene such as the polyhedrin gene by homologous recombination, or into a restriction enzyme site engineered into the desired baculovirus gene. The inserted sequences may be those which encode all or varying segments of the polyprotein, or other orfs which encode viral polypeptides. For example, the insert could encode the following numbers of amino acid segments from the polyprotein: amino acids 1-1078; amino acids 332-662; amino acids 406-662; amino acids 156-328, and amino acids 199-328.

The signals for post-translational modifications, such as signal peptide cleavage, proteolytic cleavage, and phosphorylation, appear to be recognized by insect cells. The signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells. Examples of the signal sequences from vertebrate cells which are effective in invertebrate cells are known in the art, for example, the human interleukin-2 signal (IL2_(S)) which signals for secretion from the cell, is recognized and properly removed in insect cells.

Transformation may be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus and transducing a host cell with the virus, and by direct uptake of the polynucleotide. The transformation procedure used depends upon the host to be transformed. Bacterial transformation by direct uptake generally employs treatment with calcium or rubidium chloride (Cohen, Proc Nat Acad Sci USA (1972) 69:2110; T. Maniatis et at, "Molecular Cloning; A Laboratory Manual" (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1982). Yeast transformation by direct uptake may be carried out using the method of Hinnen et al, Proc Nat Acad Sci USA (1978) 75:1929. Mammalian transformations by direct uptake may be conducted using the calcium phosphate precipitation method of Graham and Van der Eb, Virol (1978) 52:546, or the various known modifications thereof. Other methods for introducing recombinant polynucleotides into cells, particularly into mammalian cells, include dextran-mediated transfection, calcium phosphate mediated transfection, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the polynucleotides into nuclei.

Vector construction employs techniques which are known in the art. Site-specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions which generally are specified by the manufacturer of these commercially available enzymes. In general, about 1 μg of plasmid or DNA sequence is cleaved by 1 unit of enzyme in about 20 μL buffer solution by incubation for 1-2 hr at 37° C. After incubation with the restriction enzyme, protein is removed by phenol/chloroform extraction and the DNA recovered by precipitation with ethanol. The cleaved fragments may be separated using polyacrylamide or agarose gel electrophoresis techniques, according to the general procedures described in Meth Enzymol (1980) 65:499-560.

Sticky-ended cleavage fragments may be blunt ended using E. coli DNA polymerase I (Klenow fragment) with the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with S1 nuclease may also be used, resulting in the hydrolysis of any single stranded DNA portions.

Ligations are carried out under standard buffer and temperature conditions using T4 DNA ligase and ATP; sticky end ligations require less ATP and less ligase than blunt end ligations. When vector fragments are used as part of a ligation mixture, the vector fragment is often treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase to remove the 5'-phosphate, thus preventing religation of the vector. Alternatively, restriction enzyme digestion of unwanted fragments can be used to prevent ligation.

Ligation mixtures are transformed into suitable cloning hosts, such as E. coli, and successful transformants selected using the markers incorporated (e.g., antibiotic resistance), and screened for the correct construction.

Synthetic oligonucleotides may be prepared using an automated oligonucleotide synthesizer as described by Warner, DNA (1984) 3:401. If desired, the synthetic strands may be labeled with ³² P by treatment with polynucleotide kinase in the presence of ³² P-ATP under standard reaction conditions.

DNA sequences, including those isolated from cDNA libraries, may be modified by known techniques, for example by site directed mutagenesis (see e.g., Zoller, Nuc Acids Res (1982) 10:6487). Briefly, the DNA to be modified is packaged into phage as a single stranded sequence, and convened to a double stranded DNA with DNA polymerase, using as a primer a synthetic oligonucleotide complementary to the portion of the DNA to be modified, where the desired modification is included in the primer sequence. The resulting double stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria which contain copies of each strand of the phage are plated in agar to obtain plaques. Theoretically, 50% of the new plaques contain phage having the mutated sequence, and the remaining 50% have the original sequence. Replicates of the plaques are hybridized to labeled synthetic probe at temperatures and conditions which permit hybridization with the correct strand, but not with the unmodified sequence. The sequences which have been identified by hybridization are recovered and cloned.

DNA libraries may be probed using the procedure of Grunstein and Hogness Proc Nat Acad Sci USA (1975) 73:3961. Briefly, in this procedure the DNA to be probed is immobilized on nitrocellulose filters, denatured, and prehybridized with a buffer containing 0-50% formamide, 0.75M NaCl, 75 mM Na citrate, 0.02% (wt/v) each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll®, 50 mM NaH₂ PO₄ (pH 6.5), 0.1% SDS, and 100 μg/mL carrier denatured DNA. The percentage of formamide in the buffer, as well as the time and temperature conditions of the prehybridization and subsequent hybridization steps depend on the stringency required. Oligomeric probes which require lower stringency conditions are generally used with low percentages of formamide, lower temperatures, and longer hybridization times. Probes containing more than 30 or 40 nucleotides, such as those derived from cDNA or genomic sequences generally employ higher temperatures, e.g., about 40°-42° C., and a high percentage formamide, e.g., 50%. Following prehybridization, 5'-³² P-labeled oligonucleotide probe is added to the buffer, and the filters are incubated in this mixture under hybridization conditions. After washing, the treated filters are subjected to autoradiography to show the location of the hybridized probe; DNA in corresponding locations on the original agar plates is used as the source of the desired DNA.

For routine vector constructions, ligation mixtures are transformed into E. coli strain HB101 or other suitable hosts, and successful transformants selected by antibiotic resistance or other markers. Plasmids from the transformants are then prepared according to the method of Clewell et al, Proc Nat Acad Sci USA (1969) 62:1159, usually following chloramphenicol amplification (Clewell, J Bacteriol (1972) 110:667). The DNA is isolated and analyzed, usually by restriction enzyme analysis and/or sequencing. Sequencing may be performed by the dideoxy method of Sanger et at, Proc Nat Acad Sci USA (1977) 74:5463, as further described by Messing et at, Nuc Acids Res (1981) 9:309, or by the method of Maxam et at, Meth Enzymol (1980) 65:499. Problems with band compression, which are sometimes observed in GC-rich regions, were overcome by use of T-deazoguanosine according to Barr et al, Biotechniques (1986) 4:428.

The enzyme-linked immunosorbent assay (ELISA) can be used to measure either antigen or antibody concentrations. This method depends upon conjugation of an enzyme to either an antigen or an antibody, and uses the bound enzyme activity as a quantitative label. To measure antibody, the known antigen is fixed to a solid phase (e.g., a microtiter dish, plastic cup, dipstick, plastic bead, or the like), incubated with test serum dilutions, washed, incubated with anti-immunoglobulin labeled with an enzyme, and washed again. Enzymes suitable for labeling are known in the art, and include, for example, horseradish peroxidase (HRP). Enzyme activity bound to the solid phase is usually measured by adding a specific substrate, and determining product formation or substrate utilization colorimetrically. The enzyme activity bound is a direct function of the amount of antibody bound.

To measure antigen, a known specific antibody is fixed to the solid phase, the test material containing antigen is added, after an incubation the solid phase is washed, and a second enzyme-labeled antibody is added. After washing, substrate is added, and enzyme activity is measured colorimetrically, and related to antigen concentration.

Proteases of the invention may be assayed for activity by cleaving a substrate which provides detectable cleavage products. As the HCV protease is believed to cleave itself from the genomic polyprotein, one can employ this autocatalytic activity both to assay expression of the protein and determine activity. For example, of the protease is joined to its fusion partner so that the HCV protease N-terminal cleavage signal (Arg--Arg) is included, the expression product will cleave itself into fusion partner and active HCV protease. One may then assay the products, for example by western blot, to verify that the proteins produced correspond in size to the separate fusion partner and protease proteins. It is presently preferred to employ small peptide p-nitrophenyl esters or methylcoumarins, as cleavage may then be followed by spectrophotometric or fluorescent assays. Following the method described by E. D. Matayoshi et at, Science (1990) 247:231-35, one may attach a fluorescent label to one end of the substrate and a quenching molecule to the other end: cleavage is then determined by measuring the resulting increase in fluorescence. If a suitable enzyme or antigen has been employed as the fusion partner, the quantity of protein produced may easily be determined. Alternatively, one may exclude the HCV protease N-terminal cleavage signal (preventing self-cleavage) and add a separate cleavage substrate, such as a fragment of the HCV NS3 domain including the native processing signal or a synthetic analog.

In the absence of this protease activity, the HCV polyprotein should remain in its unprocessed form, and thus render the virus noninfectious. Thus, the protease is useful for assaying pharmaceutical agents for control of HCV, as compounds which inhibit the protease activity sufficiently will also inhibit vital infectivity. Such inhibitors may take the form of organic compounds, particularly compounds which mimic the cleavage site of HCV recognized by the protease. Three of the putative cleavage sites of the HCV polyprotein have the following amino acid sequences:__________________________________________________________________________Val--Ser--Ala--Arg--Arg // Gly--Arg--Glu--Ile--Leu--Leu--Gly (SEQ ID NO: 36)Ala--Ile--Leu--Arg--Arg // His--Val--Gly--Pro--Val--Ser--Cys--Gln--Arg // Gly--Tyr__________________________________________________________________________

These sites are characterized by the presence of two basic amino acids immediately before the cleavage site, and are similar to the cleavage sites recognized by other flavivirus proteases. Thus, suitable protease inhibitors may be prepared which mimic the basic/basic/small neutral motif of the HCV cleavage sites, but substituting a nonlabile linkage for the peptide bond cleaved in the natural substrate. Suitable inhibitors include peptide trifluoromethyl ketones, peptide boronic acids, peptide α-ketoesters, peptide difluoroketo compounds, peptide aldehydes, peptide diketones, and the like. For example, the peptide aidehyde N-acetyl-phenylalanyl-glycinaldehyde is a potent inhibitor of the protease papain. One may conveniently prepare and assay large mixtures of peptides using the methods disclosed in U.S. patent application Ser. No. 7/189,318, filed 2 May 1988 (published as PCT WO89/10931), incorporated herein by reference. This application teaches methods for generating mixtures of peptides up to hexapeptides having all possible amino acid sequences, and further teaches assay methods for identifying those peptides capable of binding to proteases.

Other protease inhibitors may be proteins, particularly antibodies and antibody derivatives. Recombinant expression systems may be used to generate quantities of protease sufficient for production of monoclonal antibodies (MAbs) specific for the protease. Suitable antibodies for protease inhibition will bind to the protease in a manner reducing or eliminating the enzymatic activity, typically by obscuring the active site. Suitable MAbs may be used to generate derivatives, such as Fab fragments, chimeric antibodies, altered antibodies, univalent antibodies, and single domain antibodies, using methods known in the art.

Protease inhibitors are screened using methods of the invention. In general, a substrate is employed which mimics the enzyme's natural substrate, but which provides a quantifiable signal when cleaved. The signal is preferably detectable by colorimetric or fluorometric means: however, other methods such as HPLC or silica gel chromatography, GC-MS, nuclear magnetic resonance, and the like may also be useful. After optimum substrate and enzyme concentrations are determined, a candidate protease inhibitor is added to the reaction mixture at a range of concentrations. The assay conditions ideally should resemble the conditions under which the protease is to be inhibited in vivo, i.e., under physiologic pH, temperature, ionic strength, etc. Suitable inhibitors will exhibit strong protease inhibition at concentrations which do not raise toxic side effects in the subject. Inhibitors which compete for binding to the protease active site may require concentrations equal to or greater than the substrate concentration, while inhibitors capable of binding irreversibly to the protease active site may be added in concentrations on the order of the enzyme concentration.

In a presently preferred embodiment, an inactive protease mutein is employed rather than an active enzyme. It has been found that replacing a critical residue within the active site of a protease (e.g., replacing the active site Ser of a serine protease) does not significantly alter the structure of the enzyme, and thus preserves the binding specificity. The altered enzyme still recognizes and binds to its proper substrate, but falls to effect cleavage. Thus, in one method of the invention an inactivated HCV protease is immobilized, and a mixture of candidate inhibitors added. Inhibitors that closely mimic the enzyme's preferred recognition sequence will compete more successfully for binding than other candidate inhibitors. The poorly-binding candidates may then be separated, and the identity of the strongly-binding inhibitors determined. For example, HCV protease may be prepared substituting Ala for Ser₂₂₁ (FIG. 1), providing an enzyme capable of binding the HCV protease substrate, but incapable of cleaving it. The resulting protease mutein is then bound to a solid support, for example Sephadex® beads, and packed into a column. A mixture of candidate protease inhibitors in solution is then passed through the column and fractions collected. The last fractions to elute will contain the strongest-binding compounds, and provide the preferred protease inhibitor candidates.

Protease inhibitors may be administered by a variety of methods, such as intravenously, orally, intramuscularly, intraperitoneally, bronchially, intranasally, and so forth. The preferred route of administration will depend upon the nature of the inhibitor. Inhibitors prepared as organic compounds may often be administered orally (which is generally preferred) if well absorbed. Protein-based inhibitors (such as most antibody derivatives) must generally be administered by parenteral routes.

C. EXAMPLES

The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.

EXAMPLE 1 (Preparation of HCV cDNA)

A genomic library of HCV cDNA was prepared as described in PCT WO89/046699 and U.S. Ser. No. 7/456,637. This library, ATCC accession no. 40394, has been deposited as set forth below.

EXAMPLE 2 (Expression of the Polypeptide Encoded in Clone 5-1-1.)

(A) The HCV polypeptide encoded within clone 5-1-1 (see Example 1) was expressed as a fusion polypeptide with human superoxide dismutase (SOD). This was accomplished by subcloning the clone 5-1-1 cDNA insert into the expression vector pSODCF1 (K. S. Steimer et al, J Virol (1986) 58:9; EPO 138,111) as follows. The SOD/5-1-1 expression vector was transformed into E. coli D1210 cells. These cells, named Cf1/5-1-1 in E. coli, were deposited as set forth below and have an ATCC accession no. of 67967.

First, DNA isolated from pSODCF1 was treated with BamHI and EcoRI, and the following linker was ligated into the linear DNA created by the restriction enzymes:

    __________________________________________________________________________     GAT CCT GGA ATT CTG ATA AGA CCT TAA GAC TAT TTT AA                                        (SEQ ID NO: 37)     __________________________________________________________________________

After cloning, the plasmid containing the insert was isolated.

Plasmid containing the insert was restricted with EcoRI. The HCV cDNA insert in clone 5-1-1 was excised with EcoRI, and ligated into this EcoRI linearized plasmid DNA. The DNA mixture was used to transform E. coli strain D1210 (Sadler et al, Gene (1980) 8:279). Recombinants with the 5-1-1 cDNA in the correct orientation for expressing the ORF shown in FIG. 1 were identified by restriction mapping and nucleotide sequencing.

Recombinant bacteria from one clone were induced to express the SODHCV₅₋₁₋₁ polypeptide by growing the bacteria in the presence of IPTG.

Three separate expression vectors, pcf1AB, pcf1CD, and pcf1EF were created by ligating three new linkers, AB, CD, and EF to a BamHI-EcoRI fragment derived by digesting to completion the vector pSODCF1 with EcoRI and BamHI, followed by treatment with alkaline phosphatase. The linkers were created from six oligomers, A, B, C, D, E, and F. Each oligomer was phosphorylated by treatment with kinase in the presence of ATP prior to annealing to its complementary oligomer. The sequences of the synthetic linkers were the following:

    __________________________________________________________________________     Name  DNA Sequence (5' to 3')     __________________________________________________________________________     A     GATC CTG AAT TCC TGA TAA (SEQ ID NO: 38)     B         GAC TTA AGG ACT ATT TTA A (SEQ ID NO: 39)     C     GATC CGA ATT CTG TGA TAA (SEQ ID NO: 40)     D         GCT TAA GAC ACT ATT TTA A (SEQ ID NO: 41)     E     GATC CTG GAA TTC TGA TAA (SEQ ID NO: 42)     F         GAC CTT AAG ACT ATT TTA A (SEQ ID NO: 43)     __________________________________________________________________________

Each of the three linkers destroys the original EcoRI site, and creates a new EcoRI site within the linker, but within a different reading frame. Thus, the HCV cDNA EcoRI fragments isolated from the clones, when inserted into the expression vector, were in three different reading frames.

The HCV cDNA fragments in the designated λgt11 clones were excised by digestion with EcoRI; each fragment was inserted into pcf1AB, pcf1CD, and pcf1EF. These expression constructs were then transformed into D1210 E. coli cells, the transformants cloned, and polypeptides expressed as described in part B below.

(B) Expression products of the indicated HCV cDNAs were tested for antigenicity by direct immunological screening of the colonies, using a modification of the method described in Helfman et al, Proc Nat Acad Sci USA (1983), 80:31. Briefly, the bacteria were plated onto nitrocellulose filters overlaid on ampicillin plates to give approximately 40 colonies per filter. Colonies were replica plated onto nitrocellulose filters, and the replicas were regrown overnight in the presence of 2 mM IPTG and ampicillin. The bacterial colonies were lysed by suspending the nitrocellulose filters for about 15 to 20 min in an atmosphere saturated with CHCl₃ vapor. Each filter then was placed in an individual 100 mm Petri dish containing 10 mL of 50 mM Tris HCl, pH 7.5, 150 mM NaCl, 5 mM MgClphd 2, 3% (w/v) BSA, 40 μg/mL lysozyme, and 0.1 μg/mL DNase. The plates were agitated gently for at least 8 hours at room temperature. The filters were rinsed in TBST (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 0.005% Tween® 20). After incubation, the cell residues were rinsed and incubated for one hour in TBS (TBST without Tween®) containing 10% sheep serum. The filters were then incubated with pretreated sera in TBS from individuals with NANBH, which-included 3 chimpanzees; 8 patients with chronic NANBH whose sera were positive with respect to antibodies to HCV C100-3 polypeptide (also called C100); 8 patients with chronic NANBH whose sera were negative for anti-C100 antibodies; a convalescent patient whose serum was negative for anti-C100 antibodies; and 6 patients with community-acquired NANBH, including one whose sera was strongly positive with respect to anti-C100 antibodies, and one whose sera was marginally positive with respect to anti-C100 antibodies. The sera, diluted in TBS, was pretreated by preabsorption with hSOD for at least 30 minutes at 37° C. After incubation, the filters were washed twice for 30 min with TBST. The expressed proteins which bound antibodies in the sera were labeled by incubation for 2 hours with ¹²⁵ I-labeled sheep anti-human antibody. After washing, the filters were washed twice for 30 min with TBST, dried, and autoradiographed.

EXAMPLE 3 (Cloning of Full-Length SOD-Protease Fusion Proteins) (A) pBR322-C200:

The nucleotide sequences of the HCV cDNAs used below were determined essentially as described above, except that the cDNA excised from these phages were substituted for the cDNA isolated from clone 5-1-1.

Clone C33c was isolated using a hybridization probe having the following sequence:

    __________________________________________________________________________     5' ATC AGG ACC GGG GTG AGA ACA ATT ACC ACT 3' (SEQ ID NO:     __________________________________________________________________________     44)

The sequence of the HCV cDNA in clone C33c is shown in FIG. 8, which also shows the amino acids encoded therein.

Clone 35 was isolated by screening with a synthetic polynucleotide having the sequence:

    __________________________________________________________________________     5' AAG CCA CCG TGT GCG CTA GGG CTC AAG CCC 3' (SEQ ID NO:     __________________________________________________________________________     45)

Approximately 1 in 50,000 clones hybridized with the probe. The polynucleotide and deduced amino acid sequuences for C35 are shown in FIG. 7.

Clone C31 is shown in FIG. 6, which also shows the amino acids encoded therein. A C200 cassette was constructed by ligating together a 718 bp fragment obtained by digestion of clone C33c DNA with EcoRI and HinfI, a 179 bp fragment obtained by digestion of clone C31 DNA with HinfI and BglI, and a 377 bp fragment obtained by digesting clone C35 DNA with BglI and EcoRI. The construct of ligated fragments were inserted into the EcoRI site of pBR322, yielding the plasmid pBR322-C200.

(B) C7f+C20c:

Clone 7f was isolated using a probe having the sequence:

    __________________________________________________________________________     5'-AGC AGA CAA GGG GCC TCC TAG GGT GCA TAA T-3' (SEQ ID NO:     __________________________________________________________________________     46)

The sequence of HCV cDNA in clone 7f and the amino acids encoded therein are shown in FIG. 5.

Clone C20c is isolated using a probe having the following sequence:

    ______________________________________     5'-TGC ATC AAT GGG GTG TGC TGG-3' (SEQ ID NO: 47)     ______________________________________

The sequence of HCV cDNA in clone C20c, and the amino acids encoded therein are shown in FIG. 2.

Clones 7f and C20c were digested with EcoRI and SfaNI to form 400 bp and 260 bp fragments, respectively. The fragments were then cloned into the EcoRI site of pBR322 to form the vector C7f+C20c, and transformed into HB 101 cells.

(C) C300:

Clone 8h was isolated using a probe based on the sequence of nucleotides in clone 33c. The nucleotide sequence of the probe was

    __________________________________________________________________________     5'-AGA GAC AAC CAT GAG GTC CCC GGT GTT C-3'. (SEQ ID NO:     __________________________________________________________________________     48)

The sequence of the HCV cDNA in clone 8h, and the amino acids encoded therein, are shown in FIG. 4.

Clone C26d is isolated using a probe having the following sequence:

    ______________________________________     5'-CTG TTG TGC CCC GCG GCA GCC-3' (SEQ ID NO: 49)     ______________________________________

The sequence and amino acid translation of clone C26d is shown in FIG. 3.

Clones C26d and C33c (see part A above) were transformed into the methylation minus E. coli strain GM48. Clone C26d was digested with EcoRII and DdeI to provide a 100 bp fragment. Clone C33c was digested with EcoRII and EcoRI to provide a 700 bp fragment. Clone C8h was digested with EcoRI and DdeI to provide a 208 bp fragment. These three fragments were then ligated into the EcoRI site of pBR322, and transformed into E. coli HB101, to provide the vector C300.

(D) Preparation of Full Length Clones:

A 600 bp fragment was obtained from C7f+C20c by digestion with EcoRI and NaeI, and ligated to a 945 bp NaeI/EcoRI fragment from C300, and the construct inserted into the EcoRI site of pGEM4Z (commercially available from Promega) to form the vector C7fC20cC300.

C7fC20cC300 was digested with NdeI and EcoRI to provide a 892 bp fragment, which was ligated with a 1160 bp fragment obtained by digesting C200 with NdeI and EcoRI. The resulting construct was inserted into the EcoRI site of pBR322 to provide the vector C7fC20cC300C200. Construction of this vector is illustrated schematically in FIG. 9.

EXAMPLE 4 (Preparation of E. coli Expression Vectors) (A) cf1SODp600:

This vector contains a full-length HCV protease coding sequence fused to a functional hSOD leader. The vector C7fC20cC300C200 was cleaved with EcoRI to provide a 2000 bp fragment, which was then ligated into the EcoRI site of plasmid cf1CD (Example 2A). The resulting vector encodes amino acids 1-151 of hSOD, and amino acids 946-1630 of HCV (numbered from the beginning of the polyprotein, corresponding to amino acids 1-686 in FIG. 1). The vector was labeled cf1SODp600 (sometimes referred to as P600), and was transformed into E. coli D1210 cells. These cells, ATCC accession no. 68275, were deposited as set forth below.

(B) P190:

A truncated SOD-protease fusion polynucleotide was prepared by excising a 600 bp EcoRI/NaeI fragment from C7f+C20c, blunting the fragment with Klenow fragment, ligating the blunted fragment into the Klenow-blunted EcoRI site of cf1EF (Example 2A). This polynucleotide encodes a fusion protein having amino acids 1-151 of hSOD, and amino acids 1-199 of HCV protease.

(C) P300:

A longer truncated SOD-protease fusion polynucleotide was prepared by excising an 892 bp EcoRI/NdeI fragment from C7fC20cC300, blunting the fragment with Klenow fragment, ligating the blunted fragment into the Klenow-blunted EcoRI site of cf1EF. This polynucleotide encodes a fusion protein having amino acids 1-151 of hSOD, and amino acids 1-299 of HCV protease.

(D) P500:

A longer truncated SOD-protease fusion polynucleotide was prepared by excising a 1550 bp EcoRI/EcoRI fragment from C7fC20cC300, and ligating the fragment into the EcoRI site of cf1CD to form P500. This polynucleotide encodes a fusion protein having amino acids 1-151 of hSOD, and amino acids 946-1457 of HCV protease (amino acids 1-513 in FIG. 1).

(E) FLAG/Protease Fusion

This vector contains a full-length HCV protease coding sequence fused to the FLAG sequence, Hopp et al. (1988) Biotechnology 6:1204-1210. PCR was used to produce a HCV protease gene with special restriction ends for cloning ease. Plasmid p500 was digested with EcoRI and NdeI to yield a 900 bp fragment. This fragment and two primers were used in a polymerase chain reaction to introduce a unique BglII site at amino acid 1009 and a stop codon with a SalI site at amino acid 1262 of the HCV-1, as shown in FIG. 17 of WO 90/11089, published 4 Oct. 1990. The sequence of the primers is as follows:

    __________________________________________________________________________     5' CCC GAG CAA GAT CTC CCG GCC C 3' (SEQ ID NO: 50)        and     5'CCC GGC TGC ATA AGC AGT CGA CTT GGA 3' (SEQ ID NO: 51)     __________________________________________________________________________

After 30 cycles of PCR, the reaction was digested with BglII and SalI, and the 710 bp fragment was isolated. This fragment was annealed and ligated to the following duplex:

    ______________________________________     MetAspTyrLysAspAspAspAspLysGlyArgGlu     CATGGACTACAAAGACGATGACGATAAAGGCCGGGA      CTGATGTTTCTGCTACTGCTATTTCCGGCCCTCTAGG     ______________________________________

The duplex encodes the FLAG sequence, and initiator methionine, and a 5' NcoI (SEQ ID NO:52) and SEQ ID NO:53) restriction site. The resulting NcoI/SalI fragment was ligated into a derivative of pCF1.

This construct is then transformed into E. coli D1210 cells and expression of the protease is induced by the addition of IPTG.

The FLAG sequence was fused to the HCV protease to facilitate purification. A calcium dependent monoclonal antibody, which binds to the FLAG encoded peptide, is used to purify the fusion protein without harsh eluting conditions.

EXAMPLE 5 (E. coli Expression of SOD-Protease Fusion Proteins)

(A) E. coil D1210 cells were transformed with cf1SODp600 and grown in Luria broth containing 100 μg/mL ampicillin to an OD of 0.3-0.5. IPTG was then added to a concentration of 2 mM, and the cells cultured to a final OD of 0.9 to 1.3. The cells were then lysed, and the lysate analyzed by Western blot using anti-HCV sera, as described in U.S. Ser. No. 7/456,637.

The results indicated the occurrence of cleavage, as no full length product (theoretical Mr 93 kDa) was evident on the gel. Bands corresponding to the hSOD fusion partner and the separate HCV protease appeared at relative molecular weights of about 34, 53, and 66 kDa. The 34 kDa band corresponds to the hSOD partner (about 20 kDa) with a portion of the NS3 domain, while the 53 and 66 kDa bands correspond to HCV protease with varying degrees of (possibly bacterial) processing.

(B) E. coli D1210 cells were transformed with P500 and grown in Luria broth containing 100 μg/mL ampicillin to an OD of 0.3-0.5. IPTG was then added to a concentration of 2 mM, and the cells cultured to a final OD of 0.8 to 1.0. The cells were then lysed, and the lysate analyzed as described above.

The results again indicated the occurrence of cleavage, as no full length product (theoretical Mr 73 kDa) was evident on the gel. Bands corresponding to the hSOD fusion partner and the truncated HCV protease appeared at molecular weights of about 34 and 45 kDa, respectively.

(C) E. coli D1210 cells were transformed with vectors P300 and P190 and grown as described above.

The results from P300 expression indicated the occurrence of cleavage, as no full length product (theoretical Mr 51 kDa) was evident on the gel. A band corresponding to the hSOD fusion partner appeared at a relative molecular weight of about 34. The corresponding HCV protease band was not visible, as this region of the NS3 domain is not recognized by the sera employed to detect the products. However, appearance of the hSOD band at 34 kDa rather than 51 kDa indicates that cleavage occurred.

The P190 expression product appeared only as the full (encoded) length product without cleavage, forming a band at about 40 kDa, which corresponds to the theoretical molecular weight for the uncleaved product. This may indicate that the minimum essential sequence for HCV protease extends to the region between amino acids 199 and 299.

EXAMPLE 6 (Purification of E. coli Expressed Protease)

The HCV protease and fragments expressed in Example 5 may be purified as follows:

The bacterial cells in which the polypeptide was expressed are subjected to osmotic shock and mechanical disruption, the insoluble fraction containing the protease is isolated and subjected to differential extraction with an alkaline-NaCl solution, and the polypeptide in the extract purified by chromatography on columns of S-Sepharose® and Q-Sepharose®.

The crude extract resulting from osmotic shock and mechanical disruption is prepared by suspending 1 g of the packed cells in 10 mL of a solution containing 0.02 M Tris HCl, pH 7.5, 10 mM EDTA, 20% sucrose, and incubating for 10 minutes on ice. The cells are then pelleted by centrifugation at 4,000×g for 15 min at 4° G. After the supernatant is removed, the cell pellets are resuspended in 10 mL of Buffer A1 (0.01 M Tris HCl, pH 7.5, 1 mM EDTA, 14 mM β-mercaptoethanol - "βME"), and incubated on ice for 10 minutes. The cells are again pelleted at 4,000×g for 15 minutes at 4° G. After removal of the clear supernatant (periplasmic fraction I), the cell pellets are resuspended in Buffer A1, incubated on ice for 10 minutes, and again centrifuged at 4,000×g for 15 minutes at 4° G. The clear supernatant (periplasmic fraction II) is removed, and the cell pellet resuspended in 5 mL of Buffer T2 (0.02 M Tris HCl, pH 7.5, 14 mM βME, 1 mM EDTA, 1 mM PMSF). In order to disrupt the cells, the suspension (5 mL) and 7.5 mL of Dyno-mill lead-free acid washed glass beads (0.10-0.15 mm diameter) (available from Glen-Mills, Inc.) are placed in a Falcon tube and vortexed at top speed for two minutes, followed by cooling for at least 2 min on ice. The vonexing-cooling procedure is repeated another four times. After vortexing, the slurry is filtered through a sintered glass funnel using low suction, the glass beads washed twice with Buffer A2, and the filtrate and washes combined.

The insoluble fraction of the crude extract is collected by centrifugation at 20,000×g for 15 min at 4° C., washed twice with 10 mL Buffer A2, and resuspended in 5 mL of MILLI-Q water.

A fraction containing the HCV protease is isolated from the insoluble material by adding to the suspension NaOH (2M) and NaCl (2M) to yield a final concentation of 20 mM each, vonexing the mixture for 1 minute, centrifuging it 20,000×g for 20 min at 4° C., and retaining the supernatant.

The partially purified protease is then purified by SDS-PAGE. The protease may be identified by western blot, and the band excised from the gel. The protease is then eluted from the band, and analyzed to confirm its amino acid sequence. N-terminal sequences may be analyzed using an automated amino acid sequencer, while C-terminal sequences may be analyzed by automated amino acid sequencing of a series of tryptic fragments.

EXAMPLE 7 (Preparation of Yeast Expression Vector) (A) P650 (SOD/Protease Fusion)

This vector contains HCV sequence, which includes the wild-type full-length HCV protease coding sequence, fused at the 5' end to a SOD coding sequence. Two fragments, a 441 bp EcoRI/BglII fragment from clone 11b and a 1471 bp BglII/EcoRI fragment from expression vector P500, were used to reconstruct a wild-type, full-length HCV protease coding sequence. These two fragments were ligated together with an EcoRI digested pS356 vector to produce an expression cassette. The expression cassette encodes the ADH2/GAPDH hybrid yeast promoter, human SOD, the HCV protease, and a GAPDH transcription terminator. The resulting vector was digested with BamHI and a 4052 bp fragment was isolated. This fragment was ligated to the BamHI digested pAB24 vector to produce p650. p650 expresses a polyprotein containing, from its amino terminal end, amino acids 1-154 of hSOD, an oligopeptide -Asn-Leu-Gly-Ile-Arg-, and amino acids 819 to 1458 of HCV-1, as shown in FIG. 17 of WO 90/11089, published 4 Oct. 1990.

Clone 11b was isolated from the genomic library of HCV cDNA, ATCC accession no. 40394, as described above in Example 3A, using a hybridization probe having the following sequence:

    __________________________________________________________________________     5' CAC CTA TGT TTA TAA CCA TCT CAC TCC TCT 3'. (SEQ ID NO:     __________________________________________________________________________     54)

This procedure is also described in EPO Pub. No. 318 216, Example IV.A.17.

The vector pS3EF, which is a pBR322 derivative, contains the ADH2/GAPDH hybrid yeast promoter upstream of the human superoxide dimutase gene, an adaptor, and a downstream yeast effective transcription terminator. A similar expression vector containing these control elements and the superoxide dismutase gene is described in Cousens et al. (1987) Gene 61:265, and in copending application EPO 196,056, published Oct. 1, 1986. pS3EF, however, differs from that in Cousens et al. in that the heterologous proinsulin gene and the immunoglobulin hinge are deleted, and Gln₁₅₄ of SOD is followed by an adaptor sequence which contains an EcoRI site. The sequence of the adaptor is:

    __________________________________________________________________________     5' AAT TTG GGA ATT CCA TAA TTA ATT AAG 3' (SEQ ID NO: 55)      3' AC CCT TAA GGT ATT AAT TAA TTC AGCT 5' (SEQ ID NO: 56)     __________________________________________________________________________

The EcoRI site facilitates the insertion of heterologous sequences. Once inserted into pS3EF, a SOD fusion is expressed which contains an oligopeptide that links SOD to the heterologous sequences. pS3EF is exactly the same as pS356 except that pS356 contains a different adaptor. The sequence of the adaptor is shown below:

    __________________________________________________________________________     5' AAT TTG GGA ATT CCA TAA TGA G 3' (SEQ ID NO: 57)       3' AC CCT TAA GGT ATT ACT CAG CT 5' (SEQ ID NO: 58)     __________________________________________________________________________

pS356, ATCC accession no. 67683, is deposited as set forth below.

Plasmid pAB24 is a yeast shuttle vector, which contains pBR322 sequences, the complete 2 μ sequence for DNA replication in yeast (Broach (1981) in: Molecular Biology of the Yeast Saccharomyces, Vol. 1, p. 445, Cold spring Harbor Press.) and the yeast LEU^(2d) gene derived from plasmid pC1/1, described in EPO Pub. No. 116 201. Plasmid pAB24 was constructed by digesting YEp24 with EcoRI and re-ligating the vector to remove the partial 2 micron sequences. The resulting plasmid, YEp24deltaRI, was linearized with ClaI and ligated with the complete 2 micron plasmid which had been linearized with ClaI. The resulting plasmid, pCBou, was then digested with XbaI, and the 8605 bp vector fragment was gel isolated. This isolated XbaI fragment was ligated with a 4460 bp XbaI fragment containing the LEU^(2d) gene isolated from pC1/1; the orientation of LEU^(2d) gene is in the same direction as the URA3 gene.

S. cerevisae, 2150-2-3 (pAB24-GAP-env2), accession no. 20827, is deposited with the American Type Culture Collection as set forth below. The plasmid pAB24-GAP-env2 can be recovered from the yeast cells by known techniques. The GAP-env2 expression cassette can be removed by digesting pAB24-GAP-env2 with BamHI. pAB24 is recovered by religating the vector without the BamHI insert.

EXAMPLE 8 (Yeast Expression of SOD-Protease Fusion Protein)

p650 was transformed in S. cerevisae strain JSC310, Mata, leu2, ura3-52, prb1-1122, pep4-3, prc1-407, cir°: DM15 (g418 resistance). The transformation is as described by Hinnen et al. (1978) Proc Natl Acad Sci USA 75:1929. The transformed cells were selected on ura- plates with 8% glucose. The plates were incubated at 30° C. for 4-5 days. The tranformants were further selected on leu- plates with 8% glucose putatively for high numbers of the p650 plasmid. Colonies from the leu- plates were inoculated into leu- medium with 3% glucose. These cultures were shaken at 30° C. for 2 days and then diluted 1/20 into YEPD medium with 2% glucose and shaken for 2 more days at 30° C.

S. cerevisae JSC310 contains DM15 DNA, described in EPO Pub. No. 340 986, published 8 Nov. 1989. This DM15 DNA enhances ADH2 regulated expression of heterologous proteins. pDM15, accession no. 40453, is deposited with the American Type Culture Collection as set forth below.

EXAMPLE 9 (Yeast Ubiquitin Expression of Mature HCV Protease)

Mature HCV protease is prepared by cleaving vector C7fC20cC300C200 with EcoRI to obtain a 2 Kb coding sequence, and inserting the sequence with the appropriate linkers into a ubiquitin expression vector, such as that described in WO 88/02406, published 7 Apr. 1988, or U.S. Ser. No. 7/390,599 filed 7 Aug. 1989, incorporated herein by reference. Mature HCV protease is recovered upon expression of the vector in suitable hosts, particularly yeast. Specifically, the yeast expression protocol described in Example 8 is used to express a ubiquitin/HCV protease vector.

EXAMPLE 10 (Preparation of an In-Vitro Expression Vector) (A) pGEM®-3Z/Yellow Fever Leader Vector

Four synthetic DNA fragments were annealed and ligated** together to create a EcoRI/SacI Yellow Fever leader, which was ligated to a EcoRI/SacI digested pGEM®-3Z vector from Promega®. The sequence of the four fragments are listed below:

    __________________________________________________________________________     YFK-1:     5' AAT TCG TAA ATC CTG TGT GCT AAT TGA GGT GCA TTG GTC TGC     AAA TCG AGT TGC TAG GCA ATA AAC ACA TT 3' (SEQ ID NO: 59)     YFK-2:     5' TAT TGC CTA GCA ACT CGA TTT GCA GAC CAA TGC ACC TCA ATT     AGC ACA CAG GAT TTA CG 3' (SEQ ID NO: 60)     YFK-3:     5' TGG ATT AAT TTT AAT CGT TCG TTG AGC GAT TAG CAG AGA ACT     GAC CAG AAC ATG TCT GAG CT 3' (SEQ ID NO: 61)     YFK-4:     5' CAG ACA TGT TCT GGT CAG TTC TCT GCT AAT CGC TCA ACG AAC     GAT TAA AAT TAA TCC AAA TGT GTT 3'. (SEQ ID NO: 62)     __________________________________________________________________________

For in-vitro translation of the HCV protease, the new pGEM®-3Z/Yellow Fever leader vector was digested with BamHI and blunted with Klenow.

(B) PvulI Construct from p6000

A clone p6000 was constructed from sequences available from the genomic library of HCV cDNA, ATCC accession no. 40394. The HCV encoding DNA sequence of p6000 is identical to nucleotide -275 to nucleotide 6372 of FIG. 17 of WO 90/11089, published 4 Oct. 1990. p6000 was digested with PvuII, and from the digest, a 2,864 bp fragment was isolated. This 2,864 bp fragment was ligated to the prepared pGEM®-3Z/Yellow Fever leader vector fragment, described above.

EXAMPLE 11 (In-Vitro Expression of HCV Protease) (A) Transcription

The pGEM®-3Z/Yellow Fever leader/PvulI vector was linearized with XbaI and transcribed using the materials and protocols from Promega's Riboprobe® Gemini II Core system.

(B) Translation

The RNA produced by the above protocol was translated using Promega's rabbit reticulocyte lysate, minus methionine, canine pancreatic microsomal membranes, as well as, other necessary materials and instructions from Promega.

Deposited Biological Materials:

The following materials were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Md.:

    ______________________________________     Name            Deposit Date                                Accession No.     ______________________________________     E. coli D1210,  23 Mar 1990                                68275     cf1SODp600     Cf1/5-1-1 in    11 May 1989                                67967     E. coli D1210     Bacteriophage λ-gt11                     01 Dec 1987                                40394     cDNA library     E. coli HB101, pS356                     29 Apr 1988                                67683     plasmid DNA, pDM15                     05 May 1988                                40453     S. cerevisae, 2150-2-3                     23 Dec 1986                                20827     (pAB24-GAP-env2)     ______________________________________

The above materials have been deposited with the ATCC under the accession numbers indicated. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedure. These deposits are provided as a convenience to those of skill in the art, and are not an admission that a deposit is required under 35 U.S.C. §112. The polynucleotide sequences contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the sequences described herein. A license may be required to make, use or sell the deposited materials, and no such license is granted hereby.

    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 86     (2) INFORMATION FOR SEQ ID NO:1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 202 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     ArgArgGlyArgGluIleLeuLeuGlyProAlaAspGlyMetValSer     151015     LysGlyTrpArgLeuLeuAlaProIleThrAlaTyrAlaGlnGlnThr     202530     ArgGlyLeuLeuGlyCysIleIleThrSerLeuThrGlyArgAspLys     354045     AsnGlnValGluGlyGluValGlnIleValSerThrAlaAlaGlnThr     505560     PheLeuAlaThrCysIleAsnGlyValCysTrpThrValTyrHisGly     65707580     AlaGlyThrArgThrIleAlaSerProLysGlyProValIleGlnMet     859095     TyrThrAsnValAspGlnAspLeuValGlyTrpProAlaSerGlnGly     100105110     ThrArgSerLeuThrProCysThrCysGlySerSerAspLeuTyrLeu     115120125     ValThrArgHisAlaAspValIleProValArgArgArgGlyAspSer     130135140     ArgGlySerLeuLeuSerProArgProIleSerTyrLeuLysGlySer     145150155160     SerGlyGlyProLeuLeuCysProAlaGlyHisAlaValGlyIlePhe     165170175     ArgAlaAlaValCysThrArgGlyValAlaLysAlaValAspPheIle     180185190     ProValGluAsnLeuGluThrThrMetArg     195200     (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     CysTrpThrValTyrHisGlyAlaGly     15     (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     AspGlnAspLeuGlyTrpProAlaPro     15     (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     LeuLysGlySerSerGlyGlyProLeu     15     (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     PheHisThrMetTrpHisValThrArg     15     (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     LysGluAspLeuValAlaTyrGlyGly     15     (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     ProSerGlyThrSerGlySerProIle     15     (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     PheHisThrLeuTrpHisThrThrLys     15     (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     LysGluAspArgLeuCysTyrGlyGly     15     (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     ProThrGlyThrSerGlySerProIle     15     (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     PheHisThrLeuTrpHisThrThrArg     15     (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     LysGluAspArgValThrTyrGlyGly     15     (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     ProIleGlyThrSerGlySerProIle     15     (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     PheHisThrLeuTrpHisThrThrLys     15     (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     LysGluAspArgLeuCysTyrGlyGly     15     (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     ProThrGlyThrSerGlySerProIle     15     (2) INFORMATION FOR SEQ ID NO:17:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     ThrAlaGlyHisCys     15     (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:     AsnAsnAspTyrGlyIleIle     15     (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:     GlyAspSerGlyGlySerLeu     15     (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:     ThrAlaGlyHisCys     15     (2) INFORMATION FOR SEQ ID NO:21:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:     GlyAsnAspArgAlaTrpVal     15     (2) INFORMATION FOR SEQ ID NO:22:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:     GlyAspSerGlyGlySerTrp     15     (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:     SerAlaAlaHisCys     15     (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:     AsnAsnAspIleMetLeuIle     15     (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:     GlyAspSerGlyGlyProVal     15     (2) INFORMATION FOR SEQ ID NO:26:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:     ThrAlaAlaHisCys     15     (2) INFORMATION FOR SEQ ID NO:27:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:     AsnAsnAspIleThrLeuLeu     15     (2) INFORMATION FOR SEQ ID NO:28:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:     GlyAspSerGlyGlyProLeu     15     (2) INFORMATION FOR SEQ ID NO:29:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:     ThrAlaAlaHisCys     15     (2) INFORMATION FOR SEQ ID NO:30:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:     GlyTyrAspIleAlaLeuLeu     15     (2) INFORMATION FOR SEQ ID NO:31:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:     GlyAspSerGlyGlyProLeu     15     (2) INFORMATION FOR SEQ ID NO:32:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 5 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:     ThrValTyrHisGly     15     (2) INFORMATION FOR SEQ ID NO:33:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:     SerSerAspLeuTyrLeuVal     15     (2) INFORMATION FOR SEQ ID NO:34:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 7 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:     GlySerSerGlyGlyProLeu     15     (2) INFORMATION FOR SEQ ID NO:35:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 75 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:     GlnIlePheValLysThrLeuThrGlyLysThrIleThrLeuGluVal     151015     GluSerSerAspThrIleAspAsnValLysSerLysIleGlnAspLys     202530     GluGlyIleProProAspGlnGlnArgLeuIlePheAlaGlyLysGln     354045     LeuGluAspGlyArgThrLeuSerAspTyrAsnIleGlnLysGluSer     505560     ThrLeuHisLeuValLeuArgLeuArgGlyGly     657075     (2) INFORMATION FOR SEQ ID NO:36:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 28 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:     ValSerAlaArgArgGlyArgGluIleLeuLeuGlyAlaIleLeuArg     151015     ArgHisValGlyProValSerCysGlnArgGlyTyr     2025     (2) INFORMATION FOR SEQ ID NO:37:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:     GATCCTGGAATTCTGATAAGACCTTAAGACTATTTTAA38     (2) INFORMATION FOR SEQ ID NO:38:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 19 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:     GATCCTGAATTCCTGATAA19     (2) INFORMATION FOR SEQ ID NO:39:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 19 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:     GACTTAAGGACTATTTTAA19     (2) INFORMATION FOR SEQ ID NO:40:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 19 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:     GATCCGAATTCTGTGATAA19     (2) INFORMATION FOR SEQ ID NO:41:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 19 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:     GCTTAAGACACTATTTTAA19     (2) INFORMATION FOR SEQ ID NO:42:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 19 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:     GATCCTGGAATTCTGATAA19     (2) INFORMATION FOR SEQ ID NO:43:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 19 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:     GACCTTAAGACTATTTTAA19     (2) INFORMATION FOR SEQ ID NO:44:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 30 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:     ATCAGGACCGGGGTGAGAACAATTACCACT30     (2) INFORMATION FOR SEQ ID NO:45:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 30 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:     AAGCCACCGTGTGCGCTAGGGCTCAAGCCC30     (2) INFORMATION FOR SEQ ID NO:46:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 31 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:     AGCAGACAAGGGGCCTCCTAGGGTGCATAAT31     (2) INFORMATION FOR SEQ ID NO:47:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 21 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:     TGCATCAATGGGGTGTGCTGG21     (2) INFORMATION FOR SEQ ID NO:48:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 28 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:     AGAGACAACCATGAGGTCCCCGGTGTTC28     (2) INFORMATION FOR SEQ ID NO:49:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 21 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:     CTGTTGTGCCCCGCGGCAGCC21     (2) INFORMATION FOR SEQ ID NO:50:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:     CCCGAGCAAGATCTCCCGGCCC22     (2) INFORMATION FOR SEQ ID NO:51:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:     CCCGGCTGCATAAGCAGTCGACTTGGA27     (2) INFORMATION FOR SEQ ID NO:52:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 37 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 2..37     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:     CATGGACTACAAAGACGATGACGATAAAGGCCGGGAG37     MetAspTyrLysAspAspAspAspLysGlyArgGlu     1510     (2) INFORMATION FOR SEQ ID NO:53:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 12 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:     MetAspTyrLysAspAspAspAspLysGlyArgGlu     1510     (2) INFORMATION FOR SEQ ID NO:54:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 30 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:     CACCTATGTTTATAACCATCTCACTCCTCT30     (2) INFORMATION FOR SEQ ID NO:55:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:     AATTTGGGAATTCCATAATTAATTAAG27     (2) INFORMATION FOR SEQ ID NO:56:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 27 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:     TCGACTTAATTAATTATGGAATTCCCA27     (2) INFORMATION FOR SEQ ID NO:57:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:     AATTTGGGAATTCCATAATGAG22     (2) INFORMATION FOR SEQ ID NO:58:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:     TCGACTCATTATGGAATTCCCA22     (2) INFORMATION FOR SEQ ID NO:59:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 71 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:     AATTCGTAAATCCTGTGTGCTAATTGAGGTGCATTGGTCTGCAAATCGAGTTGCTAGGCA60     ATAAACACATT71     (2) INFORMATION FOR SEQ ID NO:60:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 59 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:     TATTGCCTAGCAACTCGATTTGCAGACCAATGCACCTCAATTAGCACACAGGATTTACG59     (2) INFORMATION FOR SEQ ID NO:61:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 62 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:     TGGATTAATTTTAATCGTTCGTTGAGCGATTAGCAGAGAACTGACCAGAACATGTCTGAG60     CT62     (2) INFORMATION FOR SEQ ID NO:62:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 66 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:     CAGACATGTTCTGGTCAGTTCTCTGCTAATCGCTCAACGAACGATTAAAATTAATCCAAA60     TGTGTT66     (2) INFORMATION FOR SEQ ID NO:63:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 11 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:     TrpThrValTyrHisGlyAlaGlyThrArgThr     1510     (2) INFORMATION FOR SEQ ID NO:64:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:     LeuLysGlySerSerGlyGlyProLeu     15     (2) INFORMATION FOR SEQ ID NO:65:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 202 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:     ArgArgGlyArgGluIleLeuLeuGlyProAlaAspGlyMetValSer     151015     LysGlyTrpArgLeuLeuAlaProIleThrAlaTyrAlaGlnGlnThr     202530     ArgGlyLeuLeuGlyCysIleIleThrSerLeuThrGlyArgAspLys     354045     AsnGlnValGluGlyGluValGlnIleValSerThrAlaAlaGlnThr     505560     PheLeuAlaThrCysIleAsnGlyValCysTrpThrValTyrHisGly     65707580     AlaGlyThrArgThrIleAlaSerProLysGlyProValIleGlnMet     859095     TyrThrAsnValAspGlnAspLeuValGlyTrpProAlaSerGlnGly     100105110     ThrArgSerLeuThrProCysThrCysGlySerSerAspLeuTyrLeu     115120125     ValThrArgHisAlaAspValIleProValArgArgArgGlyAspSer     130135140     ArgGlySerLeuLeuSerProArgProIleSerTyrLeuLysGlySer     145150155160     SerGlyGlyProLeuLeuCysProAlaGlyHisAlaValGlyIlePhe     165170175     ArgAlaAlaValCysThrArgGlyValAlaLysAlaValAspPheIle     180185190     ProValGluAsnLeuGluThrThrMetArg     195200     (2) INFORMATION FOR SEQ ID NO:66:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 299 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:     GlyThrTyrValTyrAsnHisLeuThrProLeuArgAspTrpAlaHis     151015     AsnGlyLeuArgAspLeuAlaValAlaValGluProValValPheSer     202530     GlnMetGluThrLysLeuIleThrTrpGlyAlaAspThrAlaAlaCys     354045     GlyAspIleIleAsnGlyLeuProValSerAlaArgArgGlyArgGlu     505560     IleLeuLeuGlyProAlaAspGlyMetValSerLysGlyTrpArgLeu     65707580     LeuAlaProIleThrAlaTyrAlaGlnGlnThrArgGlyLeuLeuGly     859095     CysIleIleThrSerLeuThrGlyArgAspLysAsnGlnValGluGly     100105110     GluValGlnIleValSerThrAlaAlaGlnThrPheLeuAlaThrCys     115120125     IleIleAsnGlyValCysTrpThrValTyrHisGlyAlaGlyThrArg     130135140     ThrIleAlaSerProLysGlyProValIleGlnMetTyrThrAsnVal     145150155160     AspGlnAspLeuValGlyTrpProAlaSerGlnGlyThrArgSerLeu     165170175     ThrProCysThrCysGlySerSerAspLeuTyrLeuValThrArgHis     180185190     AlaAspValIleProValArgArgArgGlyAspSerArgGlySerLeu     195200205     LeuSerProArgProIleSerTyrLeuLysGlySerSerGlyGlyPro     210215220     LeuLeuCysProAlaGlyHisAlaValGlyIlePheArgAlaAlaVal     225230235240     CysThrArgGlyValAlaLysAlaValAspPheIleProValGluAsn     245250255     LeuGluThrThrMetArgSerProValPheThrAspAsnSerSerPro     260265270     ProValValProGlnSerPheGlnValAlaHisLeuHisAlaProThr     275280285     GlySerGlyLysSerThrLysValProAlaAla     290295     (2) INFORMATION FOR SEQ ID NO:67:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 199 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:     GlyThrTyrValTyrAsnHisLeuThrProLeuArgAspTrpAlaHis     151015     AsnGlyLeuArgAspLeuAlaValAlaValGluProValValPheSer     202530     GlnMetGluThrLysLeuIleThrTrpGlyAlaAspThrAlaAlaCys     354045     GlyAspIleIleAsnGlyLeuProValSerAlaArgArgGlyArgGlu     505560     IleLeuLeuGlyProAlaAspGlyMetValSerLysGlyTrpArgLeu     65707580     LeuAlaProIleThrAlaTyrAlaGlnGlnThrArgGlyLeuLeuGly     859095     CysIleIleThrSerLeuThrGlyArgAspLysAsnGlnValGluGly     100105110     GluValGlnIleValSerThrAlaAlaGlnThrPheLeuAlaThrCys     115120125     IleIleAsnGlyValCysTrpThrValTyrHisGlyAlaGlyThrArg     130135140     ThrIleAlaSerProLysGlyProValIleGlnMetTyrThrAsnVal     145150155160     AspGlnAspLeuValGlyTrpProAlaSerGlnGlyThrArgSerLeu     165170175     ThrProCysThrCysGlySerSerAspLeuTyrLeuValThrArgHis     180185190     AlaAspValIleProValArg     195     (2) INFORMATION FOR SEQ ID NO:68:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 299 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:     GlyThrTyrValTyrAsnHisLeuThrProLeuArgAspTrpAlaHis     151015     AsnGlyLeuArgAspLeuAlaValAlaValGluProValValPheSer     202530     GlnMetGluThrLysLeuIleThrTrpGlyAlaAspThrAlaAlaCys     354045     GlyAspIleIleAsnGlyLeuProValSerAlaArgArgGlyArgGlu     505560     IleLeuLeuGlyProAlaAspGlyMetValSerLysGlyTrpArgLeu     65707580     LeuAlaProIleThrAlaTyrAlaGlnGlnThrArgGlyLeuLeuGly     859095     CysIleIleThrSerLeuThrGlyArgAspLysAsnGlnValGluGly     100105110     GluValGlnIleValSerThrAlaAlaGlnThrPheLeuAlaThrCys     115120125     IleIleAsnGlyValCysTrpThrValTyrHisGlyAlaGlyThrArg     130135140     ThrIleAlaSerProLysGlyProValIleGlnMetTyrThrAsnVal     145150155160     AspGlnAspLeuValGlyTrpProAlaSerGlnGlyThrArgSerLeu     165170175     ThrProCysThrCysGlySerSerAspLeuTyrLeuValThrArgHis     180185190     AlaAspValIleProValArgArgArgGlyAspSerArgGlySerLeu     195200205     LeuSerProArgProIleSerTyrLeuLysGlySerSerGlyGlyPro     210215220     LeuLeuCysProAlaGlyHisAlaValGlyIlePheArgAlaAlaVal     225230235240     CysThrArgGlyValAlaLysAlaValAspPheIleProValGluAsn     245250255     LeuGluThrThrMetArgSerProValPheThrAspAsnSerSerPro     260265270     ProValValProGlnSerPheGlnValAlaHisLeuHisAlaProThr     275280285     GlySerGlyLysSerThrLysValProAlaAla     290295     (2) INFORMATION FOR SEQ ID NO:69:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 2064 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 7..2064     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:     ATTCGGGGCACCTATGTTTATAACCATCTCACTCCTCTTCGGGACTGG48     GlyThrTyrValTyrAsnHisLeuThrProLeuArgAspTrp     1510     GCGCACAACGGCTTGCGAGATCTGGCCGTGGCTGTAGAGCCAGTCGTC96     AlaHisAsnGlyLeuArgAspLeuAlaValAlaValGluProValVal     15202530     TTCTCCCAAATGGAGACCAAGCTCATCACGTGGGGGGCAGATACCGCC144     PheSerGlnMetGluThrLysLeuIleThrTrpGlyAlaAspThrAla     354045     GCGTGCGGTGACATCATCAACGGCTTGCCTGTTTCCGCCCGCAGGGGC192     AlaCysGlyAspIleIleAsnGlyLeuProValSerAlaArgArgGly     505560     CGGGAGATACTGCTCGGGCCAGCCGATGGAATGGTCTCCAAGGGTTGG240     ArgGluIleLeuLeuGlyProAlaAspGlyMetValSerLysGlyTrp     657075     AGGTTGCTGGCGCCCATCACGGCGTACGCCCAGCAGACAAGGGGCCTC288     ArgLeuLeuAlaProIleThrAlaTyrAlaGlnGlnThrArgGlyLeu     808590     CTAGGGTGCATAATCACCAGCCTAACTGGCCGGGACAAAAACCAAGTG336     LeuGlyCysIleIleThrSerLeuThrGlyArgAspLysAsnGlnVal     95100105110     GAGGGTGAGGTCCAGATTGTGTCAACTGCTGCCCAAACCTTCCTGGCA384     GluGlyGluValGlnIleValSerThrAlaAlaGlnThrPheLeuAla     115120125     ACGTGCATCATCAATGGGGTGTGCTGGACTGTCTACCACGGGGCCGGA432     ThrCysIleIleAsnGlyValCysTrpThrValTyrHisGlyAlaGly     130135140     ACGAGGACCATCGCGTCACCCAAGGGTCCTGTCATCCAGATGTATACC480     ThrArgThrIleAlaSerProLysGlyProValIleGlnMetTyrThr     145150155     AATGTAGACCAAGACCTTGTGGGCTGGCCCGCTTCGCAAGGTACCCGC528     AsnValAspGlnAspLeuValGlyTrpProAlaSerGlnGlyThrArg     160165170     TCATTGACACCCTGCACTTGCGGCTCCTCGGACCTTTACCTGGTCACG576     SerLeuThrProCysThrCysGlySerSerAspLeuTyrLeuValThr     175180185190     AGGCACGCCGATGTCATTCCCGTGCGCCGGCGGGGTGATAGCAGGGGC624     ArgHisAlaAspValIleProValArgArgArgGlyAspSerArgGly     195200205     AGCCTGCTGTCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGG672     SerLeuLeuSerProArgProIleSerTyrLeuLysGlySerSerGly     210215220     GGTCCGCTGTTGTGCCCCGCGGGGCACGCCGTGGGCATATTTAGGGCC720     GlyProLeuLeuCysProAlaGlyHisAlaValGlyIlePheArgAla     225230235     GCGGTGTGCACCCGTGGAGTGGCTAAGGCGGTGGACTTTATCCCTGTG768     AlaValCysThrArgGlyValAlaLysAlaValAspPheIleProVal     240245250     GAGAACCTAGAGACAACCATGAGGTCCCCGGTGTTCACGGATAACTCC816     GluAsnLeuGluThrThrMetArgSerProValPheThrAspAsnSer     255260265270     TCTCCACCAGTAGTGCCCCAGAGCTTCCAGGTGGCTCACCTCCATGCT864     SerProProValValProGlnSerPheGlnValAlaHisLeuHisAla     275280285     CCCACAGGCAGCGGCAAAAGCACCAAGGTCCCGGCTGCATATGCAGCT912     ProThrGlySerGlyLysSerThrLysValProAlaAlaTyrAlaAla     290295300     CAGGGCTATAAGGTGCTAGTACTCAACCCCTCTGTTGCTGCAACACTG960     GlnGlyTyrLysValLeuValLeuAsnProSerValAlaAlaThrLeu     305310315     GGCTTTGGTGCTTACATGTCCAAGGCTCATGGGATCGATCCTAACATC1008     GlyPheGlyAlaTyrMetSerLysAlaHisGlyIleAspProAsnIle     320325330     AGGACCGGGGTGAGAACAATTACCACTGGCAGCCCCATCACGTACTCC1056     ArgThrGlyValArgThrIleThrThrGlySerProIleThrTyrSer     335340345350     ACCTACGGCAAGTTCCTTGCCGACGGCGGGTGCTCGGGGGGCGCTTAT1104     ThrTyrGlyLysPheLeuAlaAspGlyGlyCysSerGlyGlyAlaTyr     355360365     GACATAATAATTTGTGACGAGTGCCACTCCACGGATGCCACATCCATC1152     AspIleIleIleCysAspGluCysHisSerThrAspAlaThrSerIle     370375380     TTGGGCATTGGCACTGTCCTTGACCAAGCAGAGACTGCGGGGGCGAGA1200     LeuGlyIleGlyThrValLeuAspGlnAlaGluThrAlaGlyAlaArg     385390395     CTGGTTGTGCTCGCCACCGCCACCCCTCCGGGCTCCGTCACTGTGCCC1248     LeuValValLeuAlaThrAlaThrProProGlySerValThrValPro     400405410     CATCCCAACATCGAGGAGGTTGCTCTGTCCACCACCGGAGAGATCCCT1296     HisProAsnIleGluGluValAlaLeuSerThrThrGlyGluIlePro     415420425430     TTTTACGGCAAGGCTATCCCCCTCGAAGTAATCAAGGGGGGGAGACAT1344     PheTyrGlyLysAlaIleProLeuGluValIleLysGlyGlyArgHis     435440445     CTCATCTTCTGTCATTCAAAGAAGAAGTGCGACGAACTCGCCGCAAAG1392     LeuIlePheCysHisSerLysLysLysCysAspGluLeuAlaAlaLys     450455460     CTGGTCGCATTGGGCATCAATGCCGTGGCCTACTACCGCGGTCTTGAC1440     LeuValAlaLeuGlyIleAsnAlaValAlaTyrTyrArgGlyLeuAsp     465470475     GTGTCCGTCATCCCGACCAGCGGCGATGTTGTCGTCGTGGCAACCGAT1488     ValSerValIleProThrSerGlyAspValValValValAlaThrAsp     480485490     GCCCTCATGACCGGCTATACCGGCGACTTCGACTCGGTGATAGACTGC1536     AlaLeuMetThrGlyTyrThrGlyAspPheAspSerValIleAspCys     495500505510     AATACGTGTGTCACCCAGACAGTCGATTTCAGCCTTGACCCTACCTTC1584     AsnThrCysValThrGlnThrValAspPheSerLeuAspProThrPhe     515520525     ACCATTGAGACAATCACGCTCCCCCAAGATGCTGTCTCCCGCACTCAA1632     ThrIleGluThrIleThrLeuProGlnAspAlaValSerArgThrGln     530535540     CGTCGGGGCAGGACTGGCAGGGGGAAGCCAGGCATCTACAGATTTGTG1680     ArgArgGlyArgThrGlyArgGlyLysProGlyIleTyrArgPheVal     545550555     GCACCGGGGGAGCGCCCTCCCGGCATGTTCGACTCGTCCGTCCTCTGT1728     AlaProGlyGluArgProProGlyMetPheAspSerSerValLeuCys     560565570     GAGTGCTATGACGCAGGCTGTGCTTGGTATGAGCTCACGCCCGCCGAG1776     GluCysTyrAspAlaGlyCysAlaTrpTyrGluLeuThrProAlaGlu     575580585590     ACTACAGTTAGGCTACGAGCGTACATGAACACCCCGGGGCTTCCCGTG1824     ThrThrValArgLeuArgAlaTyrMetAsnThrProGlyLeuProVal     595600605     TGCCAGGACCATCTTGAATTTTGGGAGGGCGTCTTTACAGGCCTCACT1872     CysGlnAspHisLeuGluPheTrpGluGlyValPheThrGlyLeuThr     610615620     CATATAGATGCCCACTTTCTATCCCAGACAAAGCAGAGTGGGGAGAAC1920     HisIleAspAlaHisPheLeuSerGlnThrLysGlnSerGlyGluAsn     625630635     CTTCCTTACCTGGTAGCGTACCAAGCCACCGTGTGCGCTAGGGCTCAA1968     LeuProTyrLeuValAlaTyrGlnAlaThrValCysAlaArgAlaGln     640645650     GCCCCTCCCCCATCGTGGGACCAGATGTGGAAGTGTTTGATTCGCCTC2016     AlaProProProSerTrpAspGlnMetTrpLysCysLeuIleArgLeu     655660665670     AAGCCCACCCTCCATGGGCCAACACCCCTGCTATACAGACTGGGCGCT2064     LysProThrLeuHisGlyProThrProLeuLeuTyrArgLeuGlyAla     675680685     (2) INFORMATION FOR SEQ ID NO:70:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 686 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:     GlyThrTyrValTyrAsnHisLeuThrProLeuArgAspTrpAlaHis     151015     AsnGlyLeuArgAspLeuAlaValAlaValGluProValValPheSer     202530     GlnMetGluThrLysLeuIleThrTrpGlyAlaAspThrAlaAlaCys     354045     GlyAspIleIleAsnGlyLeuProValSerAlaArgArgGlyArgGlu     505560     IleLeuLeuGlyProAlaAspGlyMetValSerLysGlyTrpArgLeu     65707580     LeuAlaProIleThrAlaTyrAlaGlnGlnThrArgGlyLeuLeuGly     859095     CysIleIleThrSerLeuThrGlyArgAspLysAsnGlnValGluGly     100105110     GluValGlnIleValSerThrAlaAlaGlnThrPheLeuAlaThrCys     115120125     IleIleAsnGlyValCysTrpThrValTyrHisGlyAlaGlyThrArg     130135140     ThrIleAlaSerProLysGlyProValIleGlnMetTyrThrAsnVal     145150155160     AspGlnAspLeuValGlyTrpProAlaSerGlnGlyThrArgSerLeu     165170175     ThrProCysThrCysGlySerSerAspLeuTyrLeuValThrArgHis     180185190     AlaAspValIleProValArgArgArgGlyAspSerArgGlySerLeu     195200205     LeuSerProArgProIleSerTyrLeuLysGlySerSerGlyGlyPro     210215220     LeuLeuCysProAlaGlyHisAlaValGlyIlePheArgAlaAlaVal     225230235240     CysThrArgGlyValAlaLysAlaValAspPheIleProValGluAsn     245250255     LeuGluThrThrMetArgSerProValPheThrAspAsnSerSerPro     260265270     ProValValProGlnSerPheGlnValAlaHisLeuHisAlaProThr     275280285     GlySerGlyLysSerThrLysValProAlaAlaTyrAlaAlaGlnGly     290295300     TyrLysValLeuValLeuAsnProSerValAlaAlaThrLeuGlyPhe     305310315320     GlyAlaTyrMetSerLysAlaHisGlyIleAspProAsnIleArgThr     325330335     GlyValArgThrIleThrThrGlySerProIleThrTyrSerThrTyr     340345350     GlyLysPheLeuAlaAspGlyGlyCysSerGlyGlyAlaTyrAspIle     355360365     IleIleCysAspGluCysHisSerThrAspAlaThrSerIleLeuGly     370375380     IleGlyThrValLeuAspGlnAlaGluThrAlaGlyAlaArgLeuVal     385390395400     ValLeuAlaThrAlaThrProProGlySerValThrValProHisPro     405410415     AsnIleGluGluValAlaLeuSerThrThrGlyGluIleProPheTyr     420425430     GlyLysAlaIleProLeuGluValIleLysGlyGlyArgHisLeuIle     435440445     PheCysHisSerLysLysLysCysAspGluLeuAlaAlaLysLeuVal     450455460     AlaLeuGlyIleAsnAlaValAlaTyrTyrArgGlyLeuAspValSer     465470475480     ValIleProThrSerGlyAspValValValValAlaThrAspAlaLeu     485490495     MetThrGlyTyrThrGlyAspPheAspSerValIleAspCysAsnThr     500505510     CysValThrGlnThrValAspPheSerLeuAspProThrPheThrIle     515520525     GluThrIleThrLeuProGlnAspAlaValSerArgThrGlnArgArg     530535540     GlyArgThrGlyArgGlyLysProGlyIleTyrArgPheValAlaPro     545550555560     GlyGluArgProProGlyMetPheAspSerSerValLeuCysGluCys     565570575     TyrAspAlaGlyCysAlaTrpTyrGluLeuThrProAlaGluThrThr     580585590     ValArgLeuArgAlaTyrMetAsnThrProGlyLeuProValCysGln     595600605     AspHisLeuGluPheTrpGluGlyValPheThrGlyLeuThrHisIle     610615620     AspAlaHisPheLeuSerGlnThrLysGlnSerGlyGluAsnLeuPro     625630635640     TyrLeuValAlaTyrGlnAlaThrValCysAlaArgAlaGlnAlaPro     645650655     ProProSerTrpAspGlnMetTrpLysCysLeuIleArgLeuLysPro     660665670     ThrLeuHisGlyProThrProLeuLeuTyrArgLeuGlyAla     675680685     (2) INFORMATION FOR SEQ ID NO:71:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 368 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..366     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:     AATTCGGAAAACCAAGTGGAGGGTGAGGTCCAGATTGTGTCAACTGCT48     AsnSerGluAsnGlnValGluGlyGluValGlnIleValSerThrAla     151015     GCCCAAACCTTCCTGGCAACGTGCATCAATGGGGTGTGCTGGACTGTC96     AlaGlnThrPheLeuAlaThrCysIleAsnGlyValCysTrpThrVal     202530     TACCACGGGGCCGGAACGAGGACCATCGCGTCACCCAAGGGTCCTGTC144     TyrHisGlyAlaGlyThrArgThrIleAlaSerProLysGlyProVal     354045     ATCCAGATGTATACCAATGTAGACCAAGACCTTGTGGGCTGGCCCGCT192     IleGlnMetTyrThrAsnValAspGlnAspLeuValGlyTrpProAla     505560     TCGCAAGGTACCCGCTCATTGACACCCTGCACTTGCGGCTCCTCGGAC240     SerGlnGlyThrArgSerLeuThrProCysThrCysGlySerSerAsp     65707580     CTTTACCTGGTCACGAGGCACGCCGATGTCATTCCCGTGCGCCGGCGG288     LeuTyrLeuValThrArgHisAlaAspValIleProValArgArgArg     859095     GGTGATAGCAGGGGCAGCCTCGTGTCGCCCCGGCCCATTTCCTACTTG336     GlyAspSerArgGlySerLeuValSerProArgProIleSerTyrLeu     100105110     AAAGGCTCCTCGGGGGGTCCGCTGCCGAATTC368     LysGlySerSerGlyGlyProLeuProAsn     115120     (2) INFORMATION FOR SEQ ID NO:72:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 122 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:     AsnSerGluAsnGlnValGluGlyGluValGlnIleValSerThrAla     151015     AlaGlnThrPheLeuAlaThrCysIleAsnGlyValCysTrpThrVal     202530     TyrHisGlyAlaGlyThrArgThrIleAlaSerProLysGlyProVal     354045     IleGlnMetTyrThrAsnValAspGlnAspLeuValGlyTrpProAla     505560     SerGlnGlyThrArgSerLeuThrProCysThrCysGlySerSerAsp     65707580     LeuTyrLeuValThrArgHisAlaAspValIleProValArgArgArg     859095     GlyAspSerArgGlySerLeuValSerProArgProIleSerTyrLeu     100105110     LysGlySerSerGlyGlyProLeuProAsn     115120     (2) INFORMATION FOR SEQ ID NO:73:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 208 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..207     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:     GAATTCGGGGGCCTGCTGTTGTGCCCCGCGGCAGCCGTGGGCATATTT48     GluPheGlyGlyLeuLeuLeuCysProAlaAlaAlaValGlyIlePhe     151015     AGGGCCGCGGTGTGCACCCGTGGAGTGGCTAAGGCGGTGGACTTTATC96     ArgAlaAlaValCysThrArgGlyValAlaLysAlaValAspPheIle     202530     CCTGTGGAGAACCTAGAGACAACCATGAGGTCCCCGGTGTTCACGGAT144     ProValGluAsnLeuGluThrThrMetArgSerProValPheThrAsp     354045     AACTCCTCTCCACCAGTAGTGCCCCAGAGCTTCCAGGTGGCTCACCTC192     AsnSerSerProProValValProGlnSerPheGlnValAlaHisLeu     505560     CATGCTCCCCGAATTC208     HisAlaProArgIle     65     (2) INFORMATION FOR SEQ ID NO:74:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 69 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:     GluPheGlyGlyLeuLeuLeuCysProAlaAlaAlaValGlyIlePhe     151015     ArgAlaAlaValCysThrArgGlyValAlaLysAlaValAspPheIle     202530     ProValGluAsnLeuGluThrThrMetArgSerProValPheThrAsp     354045     AsnSerSerProProValValProGlnSerPheGlnValAlaHisLeu     505560     HisAlaProArgIle     65     (2) INFORMATION FOR SEQ ID NO:75:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 281 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..279     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:     CCCTGCACTTGCGGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCC48     ProCysThrCysGlySerSerAspLeuTyrLeuValThrArgHisAla     151015     GATGTCATTCCCGTGCGCCGGCGGGGTGATAGCAGGGGCAGCCTGCTG96     AspValIleProValArgArgArgGlyAspSerArgGlySerLeuLeu     202530     TCGCCCCGGCCCATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTG144     SerProArgProIleSerTyrLeuLysGlySerSerGlyGlyProLeu     354045     TTGTGCCCCGCGGGGCACGCCGTGGGCATATTTAGGGCCGCGGTGTGC192     LeuCysProAlaGlyHisAlaValGlyIlePheArgAlaAlaValCys     505560     ACCCGTGGAGTGGCTAAGGCGGTGGACTTTATCCCTGTGGAGAACCTA240     ThrArgGlyValAlaLysAlaValAspPheIleProValGluAsnLeu     65707580     GAGACAACCATGAGGTCCCCGGTGTTCACGGATAACTCCTC281     GluThrThrMetArgSerProValPheThrAspAsnSer     8590     (2) INFORMATION FOR SEQ ID NO:76:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 93 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:     ProCysThrCysGlySerSerAspLeuTyrLeuValThrArgHisAla     151015     AspValIleProValArgArgArgGlyAspSerArgGlySerLeuLeu     202530     SerProArgProIleSerTyrLeuLysGlySerSerGlyGlyProLeu     354045     LeuCysProAlaGlyHisAlaValGlyIlePheArgAlaAlaValCys     505560     ThrArgGlyValAlaLysAlaValAspPheIleProValGluAsnLeu     65707580     GluThrThrMetArgSerProValPheThrAspAsnSer     8590     (2) INFORMATION FOR SEQ ID NO:77:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 416 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..414     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:     ATTCGGGGCACCTATGTTTATAACCATCTCACTCCTCTTCGGGACTGG48     IleArgGlyThrTyrValTyrAsnHisLeuThrProLeuArgAspTrp     151015     GCGCACAACGGCTTGCGAGATCTGGCCGTGGCTGTAGAGCCAGTCGTC96     AlaHisAsnGlyLeuArgAspLeuAlaValAlaValGluProValVal     202530     TTCTCCCAAATGGAGACCAAGCTCATCACGTGGGGGGCAGATACCGCC144     PheSerGlnMetGluThrLysLeuIleThrTrpGlyAlaAspThrAla     354045     GCGTGCGGTGACATCATCAACGGCTTGCCTGTTTCCGCCCGCAGGGGC192     AlaCysGlyAspIleIleAsnGlyLeuProValSerAlaArgArgGly     505560     CGGGAGATACTGCTCGGGCCAGCCGATGGAATGGTCTCCAAGGGTTGG240     ArgGluIleLeuLeuGlyProAlaAspGlyMetValSerLysGlyTrp     65707580     AGGTTGCTGGCGCCCATCACGGCGTACGCCCAGCAGACAAGGGGCCTC288     ArgLeuLeuAlaProIleThrAlaTyrAlaGlnGlnThrArgGlyLeu     859095     CTAGGGTGCATAATCACCAGCCTAACTGGCCGGGACAAAAACCAAGTG336     LeuGlyCysIleIleThrSerLeuThrGlyArgAspLysAsnGlnVal     100105110     GAGGGTGAGGTCCAGATTGTGTCAACTGCTGCCCAAACCTTCCTGGCA384     GluGlyGluValGlnIleValSerThrAlaAlaGlnThrPheLeuAla     115120125     ACGTGCATCAATGGGGTGTGCTGGCCGAATTC416     ThrCysIleAsnGlyValCysTrpProAsn     130135     (2) INFORMATION FOR SEQ ID NO:78:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 138 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:     IleArgGlyThrTyrValTyrAsnHisLeuThrProLeuArgAspTrp     151015     AlaHisAsnGlyLeuArgAspLeuAlaValAlaValGluProValVal     202530     PheSerGlnMetGluThrLysLeuIleThrTrpGlyAlaAspThrAla     354045     AlaCysGlyAspIleIleAsnGlyLeuProValSerAlaArgArgGly     505560     ArgGluIleLeuLeuGlyProAlaAspGlyMetValSerLysGlyTrp     65707580     ArgLeuLeuAlaProIleThrAlaTyrAlaGlnGlnThrArgGlyLeu     859095     LeuGlyCysIleIleThrSerLeuThrGlyArgAspLysAsnGlnVal     100105110     GluGlyGluValGlnIleValSerThrAlaAlaGlnThrPheLeuAla     115120125     ThrCysIleAsnGlyValCysTrpProAsn     130135     (2) INFORMATION FOR SEQ ID NO:79:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 308 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..306     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:     GAATTCGGGTCCGTCATCCCGACCAGCGGCGATGTTGTCGTCGTCGCA48     GluPheGlySerValIleProThrSerGlyAspValValValValAla     151015     ACCGATGCCCTCATGACCGGCTATACCGGCGACTTCGACTCGGTGATA96     ThrAspAlaLeuMetThrGlyTyrThrGlyAspPheAspSerValIle     202530     GACTGCAATACGTGTGTCACCCAGACAGTCGATTTCAGCCTTGACCCT144     AspCysAsnThrCysValThrGlnThrValAspPheSerLeuAspPro     354045     ACCTTCACCATTGAGACAATCACGCTCCCCCAAGATGCTGTCTCCCGC192     ThrPheThrIleGluThrIleThrLeuProGlnAspAlaValSerArg     505560     ACTCAACGTCGGGGCAGGACTGGCAGGGGGAAGCCAGGCATCTACAGA240     ThrGlnArgArgGlyArgThrGlyArgGlyLysProGlyIleTyrArg     65707580     TTTGTGGCACCGGGGGAGCGCCCCTCCGGCATGTTCGACTCGTCCGTC288     PheValAlaProGlyGluArgProSerGlyMetPheAspSerSerVal     859095     CTCTGTGAGTGCCCGAATTC308     LeuCysGluCysProAsn     100     (2) INFORMATION FOR SEQ ID NO:80:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 102 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:     GluPheGlySerValIleProThrSerGlyAspValValValValAla     151015     ThrAspAlaLeuMetThrGlyTyrThrGlyAspPheAspSerValIle     202530     AspCysAsnThrCysValThrGlnThrValAspPheSerLeuAspPro     354045     ThrPheThrIleGluThrIleThrLeuProGlnAspAlaValSerArg     505560     ThrGlnArgArgGlyArgThrGlyArgGlyLysProGlyIleTyrArg     65707580     PheValAlaProGlyGluArgProSerGlyMetPheAspSerSerVal     859095     LeuCysGluCysProAsn     100     (2) INFORMATION FOR SEQ ID NO:81:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 495 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..495     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:     ATTCGGTCCATTGAGACAATCACGCTCCCCCAGGATGCTGTCTCCCGC48     IleArgSerIleGluThrIleThrLeuProGlnAspAlaValSerArg     151015     ACTCAACGTCGGGGCAGGACTGGCAGGGGGAAGCCAGGCATCTACAGA96     ThrGlnArgArgGlyArgThrGlyArgGlyLysProGlyIleTyrArg     202530     TTTGTGGCACCGGGGGAGCGCCCCTCCGGCATGTTCGACTCGTCCGTC144     PheValAlaProGlyGluArgProSerGlyMetPheAspSerSerVal     354045     CTCTGTGAGTGCTATGACGCAGGCTGTGCTTGGTATGAGCTCACGCCC192     LeuCysGluCysTyrAspAlaGlyCysAlaTrpTyrGluLeuThrPro     505560     GCCGAGACTACAGTTAGGCTACGAGCGTACATGAACACCCCGGGGCTT240     AlaGluThrThrValArgLeuArgAlaTyrMetAsnThrProGlyLeu     65707580     CCCGTGTGCCAGGACCATCTTGAATTTTGGGAGGGCGTCTTTACAGGC288     ProValCysGlnAspHisLeuGluPheTrpGluGlyValPheThrGly     859095     CTCACTCATATAGATGCCCACTTTCTATCCCAGACAAAGCAGAGTGGG336     LeuThrHisIleAspAlaHisPheLeuSerGlnThrLysGlnSerGly     100105110     GAGAACCTTCCTTACCTGGTAGCGTACCAAGCCACCGTGTGCGCTAGG384     GluAsnLeuProTyrLeuValAlaTyrGlnAlaThrValCysAlaArg     115120125     GCTCAAGCCCCTCCCCCATCGTGGGACCAGATGTGGAAGTGTTTGATT432     AlaGlnAlaProProProSerTrpAspGlnMetTrpLysCysLeuIle     130135140     CGCCTCAAGCCCACCCTCCATGGGCCAACACCCCTGCTATACAGACTG480     ArgLeuLysProThrLeuHisGlyProThrProLeuLeuTyrArgLeu     145150155160     GGCGCTGCCGAATTC495     GlyAlaAlaGluPhe     165     (2) INFORMATION FOR SEQ ID NO:82:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 165 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:     IleArgSerIleGluThrIleThrLeuProGlnAspAlaValSerArg     151015     ThrGlnArgArgGlyArgThrGlyArgGlyLysProGlyIleTyrArg     202530     PheValAlaProGlyGluArgProSerGlyMetPheAspSerSerVal     354045     LeuCysGluCysTyrAspAlaGlyCysAlaTrpTyrGluLeuThrPro     505560     AlaGluThrThrValArgLeuArgAlaTyrMetAsnThrProGlyLeu     65707580     ProValCysGlnAspHisLeuGluPheTrpGluGlyValPheThrGly     859095     LeuThrHisIleAspAlaHisPheLeuSerGlnThrLysGlnSerGly     100105110     GluAsnLeuProTyrLeuValAlaTyrGlnAlaThrValCysAlaArg     115120125     AlaGlnAlaProProProSerTrpAspGlnMetTrpLysCysLeuIle     130135140     ArgLeuLysProThrLeuHisGlyProThrProLeuLeuTyrArgLeu     145150155160     GlyAlaAlaGluPhe     165     (2) INFORMATION FOR SEQ ID NO:83:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 816 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..816     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:     GAATTCGGGGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACC48     GluPheGlyAlaValAspPheIleProValGluAsnLeuGluThrThr     151015     ATGAGGTCCCCGGTGTTCACGGATAACTCCTCTCCACCAGTAGTGCCC96     MetArgSerProValPheThrAspAsnSerSerProProValValPro     202530     CAGAGCTTCCAGGTGGCTCACCTCCATGCTCCCACAGGCAGCGGCAAA144     GlnSerPheGlnValAlaHisLeuHisAlaProThrGlySerGlyLys     354045     AGCACCAAGGTCCCGGCTGCATATGCAGCTCAGGGCTATAAGGTGCTA192     SerThrLysValProAlaAlaTyrAlaAlaGlnGlyTyrLysValLeu     505560     GTACTCAACCCCTCTGTTGCTGCAACACTGGGCTTTGGTGCTTACATG240     ValLeuAsnProSerValAlaAlaThrLeuGlyPheGlyAlaTyrMet     65707580     TCCAAGGCTCATGGGATCGATCCTAACATCAGGACCGGGGTGAGAACA288     SerLysAlaHisGlyIleAspProAsnIleArgThrGlyValArgThr     859095     ATTACCACTGGCAGCCCCATCACGTACTCCACCTACGGCAAGTTCCTT336     IleThrThrGlySerProIleThrTyrSerThrTyrGlyLysPheLeu     100105110     GCCGACGGCGGGTGCTCGGGGGGCGCTTATGACATAATAATTTGTGAC384     AlaAspGlyGlyCysSerGlyGlyAlaTyrAspIleIleIleCysAsp     115120125     GAGTGCCACTCCACGGATGCCACATCCATCTTGGGCATTGGCACTGTC432     GluCysHisSerThrAspAlaThrSerIleLeuGlyIleGlyThrVal     130135140     CTTGACCAAGCAGAGACTGCGGGGGCGAGACTGGTTGTGCTCGCCACC480     LeuAspGlnAlaGluThrAlaGlyAlaArgLeuValValLeuAlaThr     145150155160     GCCACCCCTCCGGGCTCCGTCACTGTGCCCCATCCCAACATCGAGGAG528     AlaThrProProGlySerValThrValProHisProAsnIleGluGlu     165170175     GTTGCTCTGTCCACCACCGGAGAGATCCCTTTTTACGGCAAGGCTATC576     ValAlaLeuSerThrThrGlyGluIleProPheTyrGlyLysAlaIle     180185190     CCCCTCGAAGTAATCAAGGGGGGGAGACATCTCATCTTCTGTCATTCA624     ProLeuGluValIleLysGlyGlyArgHisLeuIlePheCysHisSer     195200205     AAGAAGAAGTGCGACGAACTCGCCGCAAAGCTGGTCGCATTGGGCATC672     LysLysLysCysAspGluLeuAlaAlaLysLeuValAlaLeuGlyIle     210215220     AATGCCGTGGCCTACTACCGCGGTCTTGACGTGTCCGTCATCCCGACC720     AsnAlaValAlaTyrTyrArgGlyLeuAspValSerValIleProThr     225230235240     AGCGGCGATGTTGTCGTCGTGGCAACCGATGCCCTCATGACCGGCTAT768     SerGlyAspValValValValAlaThrAspAlaLeuMetThrGlyTyr     245250255     ACCGGCGACTTCGACTCGGTGATAGACTGCAATACGTGTGCCGAATTC816     ThrGlyAspPheAspSerValIleAspCysAsnThrCysAlaGluPhe     260265270     (2) INFORMATION FOR SEQ ID NO:84:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 272 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:     GluPheGlyAlaValAspPheIleProValGluAsnLeuGluThrThr     151015     MetArgSerProValPheThrAspAsnSerSerProProValValPro     202530     GlnSerPheGlnValAlaHisLeuHisAlaProThrGlySerGlyLys     354045     SerThrLysValProAlaAlaTyrAlaAlaGlnGlyTyrLysValLeu     505560     ValLeuAsnProSerValAlaAlaThrLeuGlyPheGlyAlaTyrMet     65707580     SerLysAlaHisGlyIleAspProAsnIleArgThrGlyValArgThr     859095     IleThrThrGlySerProIleThrTyrSerThrTyrGlyLysPheLeu     100105110     AlaAspGlyGlyCysSerGlyGlyAlaTyrAspIleIleIleCysAsp     115120125     GluCysHisSerThrAspAlaThrSerIleLeuGlyIleGlyThrVal     130135140     LeuAspGlnAlaGluThrAlaGlyAlaArgLeuValValLeuAlaThr     145150155160     AlaThrProProGlySerValThrValProHisProAsnIleGluGlu     165170175     ValAlaLeuSerThrThrGlyGluIleProPheTyrGlyLysAlaIle     180185190     ProLeuGluValIleLysGlyGlyArgHisLeuIlePheCysHisSer     195200205     LysLysLysCysAspGluLeuAlaAlaLysLeuValAlaLeuGlyIle     210215220     AsnAlaValAlaTyrTyrArgGlyLeuAspValSerValIleProThr     225230235240     SerGlyAspValValValValAlaThrAspAlaLeuMetThrGlyTyr     245250255     ThrGlyAspPheAspSerValIleAspCysAsnThrCysAlaGluPhe     260265270     (2) INFORMATION FOR SEQ ID NO:85:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 2523 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 1..2523     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:     ATGGCTACAAACCCTGTTTGCGTTTTGAAGGGTGACGGCCCAGTTCAA48     MetAlaThrAsnProValCysValLeuLysGlyAspGlyProValGln     151015     GGTATTATTAACTTCGAGCAGAAGGAAAGTAATGGACCAGTGAAGGTG96     GlyIleIleAsnPheGluGlnLysGluSerAsnGlyProValLysVal     202530     TGGGGAAGCATTAAAGGACTGACTGAAGGCCTGCATGGATTCCATGTT144     TrpGlySerIleLysGlyLeuThrGluGlyLeuHisGlyPheHisVal     354045     CATGAGTTTGGAGATAATACAGCAGGCTGTACCAGTCCAGGTCCTCAC192     HisGluPheGlyAspAsnThrAlaGlyCysThrSerProGlyProHis     505560     TTTAATCCTCTATCCAGAAAACACGGTGGGCCAAAGGATGAAGAGAGG240     PheAsnProLeuSerArgLysHisGlyGlyProLysAspGluGluArg     65707580     CATGTTGGAGACTTGGGCAATGTGACTGCTGACAAAGATGGTGTGGCC288     HisValGlyAspLeuGlyAsnValThrAlaAspLysAspGlyValAla     859095     GATGTGTCTATTGAAGATTCTGTGATCTCACTCTCAGGAGACCATTGC336     AspValSerIleGluAspSerValIleSerLeuSerGlyAspHisCys     100105110     ATCATTGGCCGCACACTGGTGGTCCATGAAAAAGCAGATGACTTGGGC384     IleIleGlyArgThrLeuValValHisGluLysAlaAspAspLeuGly     115120125     AAAGGTGGAAATGAAGAAAGTACAAAGACAGGAAACGCTGGAAGTCGT432     LysGlyGlyAsnGluGluSerThrLysThrGlyAsnAlaGlySerArg     130135140     TTGGCTTGTGGTGTAATTGGGATCCGAATTCGGGGCACCTATGTTTAT480     LeuAlaCysGlyValIleGlyIleArgIleArgGlyThrTyrValTyr     145150155160     AACCATCTCACTCCTCTTCGGGACTGGGCGCACAACGGCTTGCGAGAT528     AsnHisLeuThrProLeuArgAspTrpAlaHisAsnGlyLeuArgAsp     165170175     CTGGCCGTGGCTGTAGAGCCAGTCGTCTTCTCCCAAATGGAGACCAAG576     LeuAlaValAlaValGluProValValPheSerGlnMetGluThrLys     180185190     CTCATCACGTGGGGGGCAGATACCGCCGCGTGCGGTGACATCATCAAC624     LeuIleThrTrpGlyAlaAspThrAlaAlaCysGlyAspIleIleAsn     195200205     GGCTTGCCTGTTTCCGCCCGCAGGGGCCGGGAGATACTGCTCGGGCCA672     GlyLeuProValSerAlaArgArgGlyArgGluIleLeuLeuGlyPro     210215220     GCCGATGGAATGGTGTCCAAGGGTTGGAGGTTGCTGGCGCCCATCACG720     AlaAspGlyMetValSerLysGlyTrpArgLeuLeuAlaProIleThr     225230235240     GCGTACGCCCAGCAGACAAGGGGCCTCCTAGGGTGCATAATCACCAGC768     AlaTyrAlaGlnGlnThrArgGlyLeuLeuGlyCysIleIleThrSer     245250255     CTAACTGGCCGGGACAAAAACCAAGTGGAGGGTGAGGTCCAGATTGTG816     LeuThrGlyArgAspLysAsnGlnValGluGlyGluValGlnIleVal     260265270     TCAACTGCTGCCCAAACCTTCCTGGCAACGTGCATCATCAATGGGGTG864     SerThrAlaAlaGlnThrPheLeuAlaThrCysIleIleAsnGlyVal     275280285     TGCTGGACTGTCTACCACGGGGCCGGAACGAGGACCATCGCGTCACCC912     CysTrpThrValTyrHisGlyAlaGlyThrArgThrIleAlaSerPro     290295300     AAGGGTCCTGTCATCCAGATGTATACCAATGTAGACCAAGACCTTGTG960     LysGlyProValIleGlnMetTyrThrAsnValAspGlnAspLeuVal     305310315320     GGCTGGCCCGCTTCGCAAGGTACCCGCTCATTGACACCCTGCACTTGC1008     GlyTrpProAlaSerGlnGlyThrArgSerLeuThrProCysThrCys     325330335     GGCTCCTCGGACCTTTACCTGGTCACGAGGCACGCCGATGTCATTCCC1056     GlySerSerAspLeuTyrLeuValThrArgHisAlaAspValIlePro     340345350     GTGCGCCGGCGGGGTGATAGCAGGGGCAGCCTGCTGTCGCCCCGGCCC1104     ValArgArgArgGlyAspSerArgGlySerLeuLeuSerProArgPro     355360365     ATTTCCTACTTGAAAGGCTCCTCGGGGGGTCCGCTGTTGTGCCCCGCG1152     IleSerTyrLeuLysGlySerSerGlyGlyProLeuLeuCysProAla     370375380     GGGCACGCCGTGGGCATATTTAGGGCCGCGGTGTGCACCCGTGGAGTG1200     GlyHisAlaValGlyIlePheArgAlaAlaValCysThrArgGlyVal     385390395400     GCTAAGGCGGTGGACTTTATCCCTGTGGAGAACCTAGAGACAACCATG1248     AlaLysAlaValAspPheIleProValGluAsnLeuGluThrThrMet     405410415     AGGTCCCCGGTGTTCACGGATAACTCCTCTCCACCAGTAGTGCCCCAG1296     ArgSerProValPheThrAspAsnSerSerProProValValProGln     420425430     AGCTTCCAGGTGGCTCACCTCCATGCTCCCACAGGCAGCGGCAAAAGC1344     SerPheGlnValAlaHisLeuHisAlaProThrGlySerGlyLysSer     435440445     ACCAAGGTCCCGGCTGCATATGCAGCTCAGGGCTATAAGGTGCTAGTA1392     ThrLysValProAlaAlaTyrAlaAlaGlnGlyTyrLysValLeuVal     450455460     CTCAACCCCTCTGTTGCTGCAACACTGGGCTTTGGTGCTTACATGTCC1440     LeuAsnProSerValAlaAlaThrLeuGlyPheGlyAlaTyrMetSer     465470475480     AAGGCTCATGGGATCGATCCTAACATCAGGACCGGGGTGAGAACAATT1488     LysAlaHisGlyIleAspProAsnIleArgThrGlyValArgThrIle     485490495     ACCACTGGCAGCCCCATCACGTACTCCACCTACGGCAAGTTCCTTGCC1536     ThrThrGlySerProIleThrTyrSerThrTyrGlyLysPheLeuAla     500505510     GACGGCGGGTGCTCGGGGGGCGCTTATGACATAATAATTTGTGACGAG1584     AspGlyGlyCysSerGlyGlyAlaTyrAspIleIleIleCysAspGlu     515520525     TGCCACTCCACGGATGCCACATCCATCTTGGGCATTGGCACTGTCCTT1632     CysHisSerThrAspAlaThrSerIleLeuGlyIleGlyThrValLeu     530535540     GACCAAGCAGAGACTGCGGGGGCGAGACTGGTTGTGCTCGCCACCGCC1680     AspGlnAlaGluThrAlaGlyAlaArgLeuValValLeuAlaThrAla     545550555560     ACCCCTCCGGGCTCCGTCACTGTGCCCCATCCCAACATCGAGGAGGTT1728     ThrProProGlySerValThrValProHisProAsnIleGluGluVal     565570575     GCTCTGTCCACCACCGGAGAGATCCCTTTTTACGGCAAGGCTATCCCC1776     AlaLeuSerThrThrGlyGluIleProPheTyrGlyLysAlaIlePro     580585590     CTCGAAGTAATCAAGGGGGGGAGACATCTCATCTTCTGTCATTCAAAG1824     LeuGluValIleLysGlyGlyArgHisLeuIlePheCysHisSerLys     595600605     AAGAAGTGCGACGAACTCGCCGCAAAGCTGGTCGCATTGGGCATCAAT1872     LysLysCysAspGluLeuAlaAlaLysLeuValAlaLeuGlyIleAsn     610615620     GCCGTGGCCTACTACCGCGGTCTTGACGTGTCCGTCATCCCGACCAGC1920     AlaValAlaTyrTyrArgGlyLeuAspValSerValIleProThrSer     625630635640     GGCGATGTTGTCGTCGTGGCAACCGATGCCCTCATGACCGGCTATACC1968     GlyAspValValValValAlaThrAspAlaLeuMetThrGlyTyrThr     645650655     GGCGACTTCGACTCGGTGATAGACTGCAATACGTGTGTCACCCAGACA2016     GlyAspPheAspSerValIleAspCysAsnThrCysValThrGlnThr     660665670     GTCGATTTCAGCCTTGACCCTACCTTCACCATTGAGACAATCACGCTC2064     ValAspPheSerLeuAspProThrPheThrIleGluThrIleThrLeu     675680685     CCCCAAGATGCTGTCTCCCGCACTCAACGTCGGGGCAGGACTGGCAGG2112     ProGlnAspAlaValSerArgThrGlnArgArgGlyArgThrGlyArg     690695700     GGGAAGCCAGGCATCTACAGATTTGTGGCACCGGGGGAGCGCCCTCCC2160     GlyLysProGlyIleTyrArgPheValAlaProGlyGluArgProPro     705710715720     GGCATGTTCGACTCGTCCGTCCTCTGTGAGTGCTATGACGCAGGCTGT2208     GlyMetPheAspSerSerValLeuCysGluCysTyrAspAlaGlyCys     725730735     GCTTGGTATGAGCTCACGCCCGCCGAGACTACAGTTAGGCTACGAGCG2256     AlaTrpTyrGluLeuThrProAlaGluThrThrValArgLeuArgAla     740745750     TACATGAACACCCCGGGGCTTCCCGTGTGCCAGGACCATCTTGAATTT2304     TyrMetAsnThrProGlyLeuProValCysGlnAspHisLeuGluPhe     755760765     TGGGAGGGCGTCTTTACAGGCCTCACTCATATAGATGCCCACTTTCTA2352     TrpGluGlyValPheThrGlyLeuThrHisIleAspAlaHisPheLeu     770775780     TCCCAGACAAAGCAGAGTGGGGAGAACCTTCCTTACCTGGTAGCGTAC2400     SerGlnThrLysGlnSerGlyGluAsnLeuProTyrLeuValAlaTyr     785790795800     CAAGCCACCGTGTGCGCTAGGGCTCAAGCCCCTCCCCCATCGTGGGAC2448     GlnAlaThrValCysAlaArgAlaGlnAlaProProProSerTrpAsp     805810815     CAGATGTGGAAGTGTTTGATTCGCCTCAAGCCCACCCTCCATGGGCCA2496     GlnMetTrpLysCysLeuIleArgLeuLysProThrLeuHisGlyPro     820825830     ACACCCCTGCTATACAGACTGGGCGCT2523     ThrProLeuLeuTyrArgLeuGlyAla     835840     (2) INFORMATION FOR SEQ ID NO:86:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 841 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:     MetAlaThrAsnProValCysValLeuLysGlyAspGlyProValGln     151015     GlyIleIleAsnPheGluGlnLysGluSerAsnGlyProValLysVal     202530     TrpGlySerIleLysGlyLeuThrGluGlyLeuHisGlyPheHisVal     354045     HisGluPheGlyAspAsnThrAlaGlyCysThrSerProGlyProHis     505560     PheAsnProLeuSerArgLysHisGlyGlyProLysAspGluGluArg     65707580     HisValGlyAspLeuGlyAsnValThrAlaAspLysAspGlyValAla     859095     AspValSerIleGluAspSerValIleSerLeuSerGlyAspHisCys     100105110     IleIleGlyArgThrLeuValValHisGluLysAlaAspAspLeuGly     115120125     LysGlyGlyAsnGluGluSerThrLysThrGlyAsnAlaGlySerArg     130135140     LeuAlaCysGlyValIleGlyIleArgIleArgGlyThrTyrValTyr     145150155160     AsnHisLeuThrProLeuArgAspTrpAlaHisAsnGlyLeuArgAsp     165170175     LeuAlaValAlaValGluProValValPheSerGlnMetGluThrLys     180185190     LeuIleThrTrpGlyAlaAspThrAlaAlaCysGlyAspIleIleAsn     195200205     GlyLeuProValSerAlaArgArgGlyArgGluIleLeuLeuGlyPro     210215220     AlaAspGlyMetValSerLysGlyTrpArgLeuLeuAlaProIleThr     225230235240     AlaTyrAlaGlnGlnThrArgGlyLeuLeuGlyCysIleIleThrSer     245250255     LeuThrGlyArgAspLysAsnGlnValGluGlyGluValGlnIleVal     260265270     SerThrAlaAlaGlnThrPheLeuAlaThrCysIleIleAsnGlyVal     275280285     CysTrpThrValTyrHisGlyAlaGlyThrArgThrIleAlaSerPro     290295300     LysGlyProValIleGlnMetTyrThrAsnValAspGlnAspLeuVal     305310315320     GlyTrpProAlaSerGlnGlyThrArgSerLeuThrProCysThrCys     325330335     GlySerSerAspLeuTyrLeuValThrArgHisAlaAspValIlePro     340345350     ValArgArgArgGlyAspSerArgGlySerLeuLeuSerProArgPro     355360365     IleSerTyrLeuLysGlySerSerGlyGlyProLeuLeuCysProAla     370375380     GlyHisAlaValGlyIlePheArgAlaAlaValCysThrArgGlyVal     385390395400     AlaLysAlaValAspPheIleProValGluAsnLeuGluThrThrMet     405410415     ArgSerProValPheThrAspAsnSerSerProProValValProGln     420425430     SerPheGlnValAlaHisLeuHisAlaProThrGlySerGlyLysSer     435440445     ThrLysValProAlaAlaTyrAlaAlaGlnGlyTyrLysValLeuVal     450455460     LeuAsnProSerValAlaAlaThrLeuGlyPheGlyAlaTyrMetSer     465470475480     LysAlaHisGlyIleAspProAsnIleArgThrGlyValArgThrIle     485490495     ThrThrGlySerProIleThrTyrSerThrTyrGlyLysPheLeuAla     500505510     AspGlyGlyCysSerGlyGlyAlaTyrAspIleIleIleCysAspGlu     515520525     CysHisSerThrAspAlaThrSerIleLeuGlyIleGlyThrValLeu     530535540     AspGlnAlaGluThrAlaGlyAlaArgLeuValValLeuAlaThrAla     545550555560     ThrProProGlySerValThrValProHisProAsnIleGluGluVal     565570575     AlaLeuSerThrThrGlyGluIleProPheTyrGlyLysAlaIlePro     580585590     LeuGluValIleLysGlyGlyArgHisLeuIlePheCysHisSerLys     595600605     LysLysCysAspGluLeuAlaAlaLysLeuValAlaLeuGlyIleAsn     610615620     AlaValAlaTyrTyrArgGlyLeuAspValSerValIleProThrSer     625630635640     GlyAspValValValValAlaThrAspAlaLeuMetThrGlyTyrThr     645650655     GlyAspPheAspSerValIleAspCysAsnThrCysValThrGlnThr     660665670     ValAspPheSerLeuAspProThrPheThrIleGluThrIleThrLeu     675680685     ProGlnAspAlaValSerArgThrGlnArgArgGlyArgThrGlyArg     690695700     GlyLysProGlyIleTyrArgPheValAlaProGlyGluArgProPro     705710715720     GlyMetPheAspSerSerValLeuCysGluCysTyrAspAlaGlyCys     725730735     AlaTrpTyrGluLeuThrProAlaGluThrThrValArgLeuArgAla     740745750     TyrMetAsnThrProGlyLeuProValCysGlnAspHisLeuGluPhe     755760765     TrpGluGlyValPheThrGlyLeuThrHisIleAspAlaHisPheLeu     770775780     SerGlnThrLysGlnSerGlyGluAsnLeuProTyrLeuValAlaTyr     785790795800     GlnAlaThrValCysAlaArgAlaGlnAlaProProProSerTrpAsp     805810815     GlnMetTrpLysCysLeuIleArgLeuLysProThrLeuHisGlyPro     820825830     ThrProLeuLeuTyrArgLeuGlyAla     835840     __________________________________________________________________________ 

What is claimed:
 1. A method for assaying compounds for activity against hepatitis C virus comprising the steps of:providing an NS3 domain hepatitis C virus protease or active NS3 domain hepatitis C virus protease truncation analog and a NS3 domain hepatitis C virus protease peptide substrate; contacting said protease or protease truncation analog with a candidate inhibitory compound in the presence of said peptide substrate; and measuring the inhibition of the proteolytic activity of said NS3 domain hepatitis C virus protease or protease truncation analog.
 2. The method of claim 1, wherein said NS3 domain hepatitis C virus protease peptide substrate comprises a covalently-linked marker compound.
 3. The method of claim 1, wherein said NS3 domain hepatitis C virus protease or active NS3 domain hepatitis C virus protease truncation analog has a partial internal amino acid sequence comprising SEQ ID NO:63.
 4. The method of claim 1, wherein said NS3 domain hepatitis C virus protease or active NS3 domain hepatitis C virus protease truncation analog has a partial internal sequence comprising SEQ ID NO:64.
 5. The method of claim 1, wherein said NS3 domain hepatitis C virus protease or active NS3 domain hepatitus C virus protease truncation analog has an amino acid sequence comprising SEQ ID NO:65. 